U.S. patent number 7,989,417 [Application Number 10/542,396] was granted by the patent office on 2011-08-02 for linkers for radiopharmaceutical compounds.
This patent grant is currently assigned to Bracco Imaging S.p.A.. Invention is credited to Christoph De Haen, Adrian D. Nunn, Rolf E. Swenson.
United States Patent |
7,989,417 |
De Haen , et al. |
August 2, 2011 |
Linkers for radiopharmaceutical compounds
Abstract
A new and improved method for extending the half life of
pharmaceutical compounds for use in diagnostic imaging or therapy
uses a novel linker to attach a diagnostic or therapeutic moiety to
a targeting peptide or another diagnostic or therapeutic moiety.
The resulting compound may have the general formula M-N-O-P-Q,
wherein M is the diagnostic or therapeutic moiety, N-O-P is the
linker of the present invention, and Q is the targeting peptide. In
another embodiment the compounds may have the formula M-N-O-P-M,
wherein M is independently a diagnostic or therapeutic moiety and
N-O-P is the linker of the invention. Methods for imaging or
treating a patient using the compounds of the invention are also
provided. Methods and kits for preparing a diagnostic imaging agent
from the compound are further provided. Methods for radiotherapy of
a patient using the compounds are further provided, as are methods
for preparing a radiotherapeutic agent from the compounds.
Inventors: |
De Haen; Christoph (Borromeo,
IT), Nunn; Adrian D. (Lamberville, NJ), Swenson;
Rolf E. (Princeton, NJ) |
Assignee: |
Bracco Imaging S.p.A. (Milan,
IT)
|
Family
ID: |
32713508 |
Appl.
No.: |
10/542,396 |
Filed: |
December 24, 2003 |
PCT
Filed: |
December 24, 2003 |
PCT No.: |
PCT/US03/41656 |
371(c)(1),(2),(4) Date: |
February 27, 2006 |
PCT
Pub. No.: |
WO2004/062574 |
PCT
Pub. Date: |
July 29, 2004 |
Prior Publication Data
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|
|
Document
Identifier |
Publication Date |
|
US 20060241018 A1 |
Oct 26, 2006 |
|
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
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60439722 |
Jan 13, 2003 |
|
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Current U.S.
Class: |
514/5.9; 514/9.7;
514/10.6; 530/328 |
Current CPC
Class: |
C07J
41/00 (20130101); A61K 51/0493 (20130101); A61K
51/088 (20130101); A61P 35/00 (20180101); A61P
3/10 (20180101); C07K 7/23 (20130101); C07K
14/62 (20130101); A61K 47/64 (20170801) |
Current International
Class: |
A61K
38/28 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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WO91/01144 |
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Feb 1991 |
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WO |
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WO96/03427 |
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Feb 1996 |
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WO |
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WO 98/02192 |
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Jan 1998 |
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WO |
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99/55376 |
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Nov 1999 |
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WO |
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01/09163 |
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Feb 2001 |
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WO |
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PCT/EP01/01971 |
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Feb 2001 |
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WO |
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01/64708 |
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Sep 2001 |
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WO |
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WO 01/64708 |
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Sep 2001 |
|
WO |
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WO 01/76531 |
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Oct 2001 |
|
WO |
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02/087597 |
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Nov 2002 |
|
WO |
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|
Primary Examiner: Tate; Christopher R.
Assistant Examiner: Teller; Roy
Attorney, Agent or Firm: Kramer Levin Naftalis & Frankel
LLP
Parent Case Text
This application is the national stage filing of corresponding
international application number PCT/US2003/041656, filed Dec. 24,
2003, which claims priority to and benefit of U.S. provisional
application No. 60/439,722, filed Jan. 13, 2003, all of which are
hereby incorporated by reference.
Claims
What is claimed is:
1. A compound of the general formula: M-N-O-P-Q wherein M is a
metal chelator; N is absent, an alpha amino acid, a substituted
bile acid or other linking group; O is an alpha amino acid or a
substituted bile acid; and P is absent, an alpha amino acid, a
substituted bile acid or other linking group; and Q is a targeting
peptide, and wherein at least one of N, O or P is a substituted
bile acid.
2. The compound of claim 1, wherein Q is a peptide hormone selected
from the group consisting of luetinising hormone releasing hormone
(LHRH), insulin, oxytosin, somatostatin, neuro kinin-1 (NK-1),
vasoactive intestinal peptide (VIP), Substance P, neuropeptide Y
(NPY), endothelin A, endothelin B, bradykinin, interleukin-1,
epidural growth factor (EGF), cholecystokinin (CCK), galanan, MSH,
Lanreotide, Octreotide, Maltose, arginine-vasopressin and analogues
and derivatives thereof.
3. The compound of claim 1, wherein the substituted bile acid is
selected from the group consisting of:
(3.beta.,5.beta.)-3-aminocholan-24-oic acid;
(3.beta.,5.beta.,12.alpha.)-3-amino-12-hydroxycholan-24-oic acid;
(3.beta.,5.beta.,7.alpha.,12.alpha.)-3-amino-7,12-dihydroxycholan-24-oic
acid;
Lys(3,6,9)-trioxaundecane-1,11-dicarbonyl-3,7-dideoxy-3-aminocholic
acid);
(3.beta.,5.beta.,7.alpha.)-3-amino-7-hydroxy-12-oxocholan-24-oic
acid; and (3.beta.,5.beta.,7.alpha.)-3-amino-7-hydroxycholan-24-oic
acid.
4. A compound of the general formula: M-N-O-P-Q wherein M is a
metal chelator; N is absent, an alpha amino acid, a substituted
bile acid or other linking group; O is an alpha amino acid or a
substituted bile acid; and P is absent, an alpha amino acid, a
substituted bile acid or other linking group; and Q is a
luetinising hormone releasing hormone (LHRH) peptide or an
analogue, and wherein at least one of N, O or P is a substituted
bile acid.
5. A compound of claim 4, wherein Q is a luetinising hormone
releasing hormone (LHRH) analogue of the formula:
PGlu-His-Trp-W-Tyr-DLys-X-Y-Pro-Z, wherein W=Ser, NMeSer, or Thr;
X=Leu, NMeLeu, t-ButylGly; Y=Arg, Arg(Et2), Cit, Lys(isopropyl);
and Z=Gly-NH.sub.2, NHEthyl, Azagly-NH.sub.2.
6. A compound of the general formula:
Glu-His-Trp-W-Tyr-DLys(M-N-O-P)-X-Y-Pro-Z, wherein M is a chelator;
N is absent, an alpha amino acid, a substituted bile acid or other
linking group; O is an alpha amino acid or a substituted bile acid;
and P is absent, an alpha amino acid, a substituted bile acid or
other linking group, W=Ser, NMeSer, or Thr. X=Leu, NMeLeu,
t-ButylGly. Y=Arg, Arg(Et2), Cit, Lys(isopropyl). Z=Gly-NH.sub.2,
NHEthyl, Azagly-NH.sub.2. wherein at least one of N, O or P is a
substituted bile amino acid.
7. The compound of claim 5, wherein M is 1, 4, 7,
10-tetraazacyclotetradecane-1, 4, 7, 10-tetraacetic acid
(DOTA)-Gly.
8. The compound of claim 4 or 5, wherein the substituted bile acid
is selected from the group consisting of:
(3.beta.,3.beta.)-3-aminocholan-24-oic acid;
(3.beta.,5.beta.,12.alpha.)-3-amino-12-hydroxycholan-24-oic acid;
(3.beta.,5.beta.,7.alpha.,12.alpha.)-3-amino-7,12-dihydroxycholan-24-oic
acid;
Lys-(3,6,9)-trioxaundecane-1,11-dicarbonyl-3,7-dideoxy-3-aminocholi-
c acid);
(3.beta.,5.beta.,7.alpha.)-3-amino-7-hydroxy-12-oxocholan-24-oic
acid; and (3.beta.,5.beta.,7.alpha.)-3-amino-7-hydroxycholan-24-oic
acid.
9. A method of imaging comprising the steps of: administering to a
patient a diagnostic imaging agent comprising the compound of claim
1 wherein the chelator is complexed with a diagnostic radionuclide
or a paramagnetic metal, and imaging said patient.
10. A method of radiotherapy comprising the steps of: administering
to a patient a radiotherapeutic agent comprising the compound of
claim 1 wherein the chelator is complexed with a therapeutic
radionuclide, and imaging said patient.
11. A method of imaging comprising the steps of: administering to a
patient a diagnostic imaging agent comprising the compound of claim
4 wherein the chelator is complexed with a diagnostic radionuclide
or a paramagnetic metal, and imaging said patient.
12. A method of radiotherapy comprising the steps of: administering
to a patient a radiotherapeutic agent comprising the compound of
claim 4 wherein the chelator is complexed with a therapeutic
radionuclide, and imaging said patient.
13. A method of imaging comprising the steps of: administering to a
patient a diagnostic imaging agent comprising the compound of claim
6 wherein the chelator is complexed with a diagnostic radionuclide
or a paramagnetic metal, and imaging said patient.
14. A method of radiotherapy comprising the steps of: administering
to a patient a radiotherapeutic agent comprising the compound of
claim 6 wherein the chelator is complexed with a therapeutic
radionuclide, and imaging said patient.
Description
FIELD OF THE INVENTION
This invention relates to new and improved methods for improving
the half life of pharmaceutical compounds which are useful as
diagnostic or therapeutic agents by using novel linkers to attach a
diagnostic or therapeutic moiety to a targeting moiety, or to
attach two diagnostic or therapeutic moieties to each other, or to
attach one diagnostic moiety to a therapeutic moiety.
BACKGROUND OF THE INVENTION
The use of pharmaceuticals (e.g., as diagnostic imaging agents,
therapeutic agents, etc.) to detect and treat cancer is well known.
In more recent years, the discovery of site-directed
radiopharmaceuticals for cancer detection and/or treatment has
gained popularity and continues to grow as the medical profession
better appreciates the specificity, efficacy and utility of such
compounds.
These newer radiopharmaceutical agents typically consist of a
targeting agent connected to a metal chelator, which can be
chelated to (e.g., complexed with) a diagnostic metal radionuclide
such as, for example, technetium or indium, or a therapeutic metal
radionuclide such as, for example, lutetium, yttrium, or rhenium.
The role of the metal chelator is to hold (i.e., chelate) the metal
radionuclide as the radiopharmaceutical agent is delivered to the
desired site. A metal chelator which does not bind strongly to the
metal radionuclide would render the radiopharmaceutical agent
ineffective for its desired use since the metal radionuclide would
therefore not reach its desired site. Thus, further research and
development led to the discovery of metal chelators, such as that
reported in U.S. Pat. No. 5,662,885 to Pollak et. al., hereby
incorporated by reference, which exhibited strong binding affinity
for metal radionuclides and the ability to conjugate with the
targeting agent. Subsequently, the concept of using a "spacer" to
create a physical separation between the metal chelator and the
targeting agent was further introduced, for example in U.S. Pat.
No. 5,976,495 to Pollak et. al., hereby incorporated by
reference.
The role of the targeting agent, by virtue of its affinity for
certain binding sites, is to direct the diagnostic or therapeutic
agent, such as, for example, a radiopharmaceutical agent containing
the metal radionuclide, to the desired site for detection or
treatment. Typically, the targeting agent may include a protein, a
peptide, or other macromolecule which exhibits a specific affinity
for a given receptor. Other known targeting agents include
monoclonal antibodies (MAbs), antibody fragments (F.sub.ab and
(F.sub.ab).sub.2), and receptor-avid peptides. Donald J. Buchsbaum,
"Cancer Therapy with Radiolabeled Antibodies; Pharmacokinetics of
Antibodies and Their Radiolabels; Experimental Radioimmunotherapy
and Methods to Increase Therapeutic Efficacy," CRC Press, Boca
Raton, Chapter 10, pp. 115-140, (1995); Fischman, et al. "A Ticket
to Ride: Peptide Radiopharmaceuticals," The Journal of Nuclear
Medicine, vol. 34, No. 12, (December 1993):
In designing an effective compound for use as a diagnostic or
therapeutic agent for cancer, it is important that the drug have
appropriate in vivo targeting and pharmacokinetic properties. For
example, it is preferable that for a radiopharmaceutical, the
radiolabeled peptide has high specific uptake by the targeted
cells. In addition, it is also preferred that once the radionuclide
localizes at a targeted site, it remains there for a desired amount
of time to deliver a highly localized radiation dose to the
site.
Moreover, developing radiolabeled peptides that are cleared
efficiently from normal tissues is also an important factor for
radiopharmaceutical agents. When biomolecules (e.g., MAb, F.sub.ab
or peptides) labeled with metallic radionuclides (via a chelate
conjugation), are administered to an animal such as a human, a
large percentage of the metallic radionuclide (in some chemical
form) can become "trapped" in either the kidney or liver parenchyma
(i.e., is not excreted into the urine or bile). Duncan et al.;
Indium-111-Diethylenetriaminepentaacetic Acid-Octreotide Is
Delivered in Vivo to Pancreatic, Tumor Cell, Renal, and Hepatocyte
Lysosomes, Cancer Research 57, pp. 659-671, (Feb. 15, 1997). For
the smaller radiolabeled biomolecules (i.e., peptides or F.sub.ab),
the major route of clearance of activity is through the kidneys
which can also retain high levels of the radioactive metal (i.e.,
normally >10-15% of the injected dose). Retention of metal
radionuclides in the kidney or liver is undesirable.
Conversely, clearance of a diagnostic or therapeutic agent from the
blood stream too quickly by the kidney is also undesirable if
longer diagnostic imaging or high tumor uptake for radiotherapy is
needed. The retention of the radiopharmaceutical compound in the
patient is often measured in terms of half life (i.e., the time it
takes for one half of the administered dosage to be cleared from
the patient). A radiopharmaceutical compound with a shorter half
life indicates that it is cleared from the patient at a faster rate
than a radiopharmaceutical compound with a longer half life.
A new and improved method for improving the half life of
pharmaceutical compounds has now been found leading to improved
diagnostic and therapeutic agents.
SUMMARY OF THE INVENTION
In an exemplary embodiment of the present invention, there is
provided a new and improved method for improving the half life of a
pharmaceutical compound for diagnostic or therapeutic use, as well
as compounds which exhibit such an improved half life. In one
embodiment, a diagnostic or therapeutic agent (such as, for
example, a chemical moiety capable of complexing a medically useful
metal ion or radionuclide (metal chelator) or an optical label) is
attached to a targeting peptide by a linker or spacer group of the
present invention. Alternatively, a radioactive halogen is attached
to a targeting peptide by a linker or spacer group of the present
invention.
As a result, compounds of the invention may generally have the
formula: M-N-O-P-Q wherein M is the diagnostic or therapeutic
agent, N-O-P is the linker, and Q is the targeting moiety. In one
embodiment, M may be a metal chelator in the form complexed with a
metal radionuclide or not. Alternatively, M may be a radioactive
halogen instead of a metal chelator.
The metal chelator M may be any of the metal chelators known in the
art for complexing with a medically useful metal ion or
radionuclide. Preferred chelators include DTPA, DOTA, DO3A,
HP-DO3A, EDTA, TETA, EHPG, HBED, NOTA, DOTMA, TETMA, PDTA, TTHA,
LICAM, MECAM, or peptide chelators, such as, for example, those
discussed herein. The metal chelator may or may not be complexed
with a metal radionuclide, and may include an optional spacer such
as a single amino acid. Preferred metal radionuclides for
scintigraphy or radiotherapy include .sup.99mTc, .sup.51Cr,
.sup.67Ga, .sup.68Ga, .sup.47Sc, .sup.51Cr, .sup.167Tm, .sup.141Ce,
.sup.111In, .sup.168Yb, .sup.175Yb, .sup.140La, .sup.90Y, .sup.88Y,
.sup.153Sm, .sup.166Ho, .sup.165Dy, .sup.166Dy, .sup.62Cu,
.sup.64Cu, .sup.67Cu, .sup.97Ru, .sup.103Ru, .sup.186Re,
.sup.188Re, .sup.203Pb, .sup.211Bi, .sup.212Bi, .sup.213Bi,
.sup.214Bi, .sup.105Rh, .sup.109Pd, .sup.117mSn, .sup.149Pm,
.sup.161Tb, .sup.177Lu, .sup.198Au, and .sup.199Au. The choice of
metal will be determined based on the desired therapeutic or
diagnostic application. For example, for diagnostic purposes the
preferred radionuclides include .sup.64Cu, .sup.67Ga, .sup.68Ga,
.sup.99mTc, and .sup.111In with .sup.99mTc, and .sup.111In being
particularly preferred. For therapeutic purposes, the preferred
radionuclides include .sup.64Cu, .sup.90Y, .sup.105Rh and .sup.90Y,
.sup.111In, .sup.117mSn, .sup.149Pm, .sup.153Sm, .sup.161Tb,
.sup.166Dy, .sup.166Ho, .sup.175Yb, .sup.177Lu, .sup.186/188Re and
.sup.199Au, with .sup.177Lu, .sup.90Y, .sup.186Re and .sup.188Re
being particularly preferred. A most preferred chelator used in
compounds of the invention is 1-substituted
4,7,10-tricarboxymethyl-1,4,7,10 tetraazacyclododecane triacetic
acid (DO3A).
Where M is a radioactive halogen, preferred radionuclides such as
.sup.18F, .sup.124I, .sup.125I, .sup.131I, .sup.123I, .sup.77Br,
and .sup.76Br may be used.
Where M is a diagnostic moiety, preferred diagnostic moieties
include, for example, agents which enable detection of the
compounds by such techniques as x-ray, magnetic resonance imaging,
ultrasound, fluorescence and other optical imaging methodologies. A
particularly preferred diagnostic moiety is a photolabel.
Where M is a therapeutic moiety, preferred compounds of the present
invention can incorporate, for example, therapeutic moieties such
as antibiotics, hormones, enzymes, antibodies, growth factors,
etc.
The linker N-O-P contains at least one substituted bile acid.
The targeting peptide Q is any peptide, equivalent, derivative or
analogue thereof which has binding affinity for a particular site.
The targeting peptide may be a peptide that binds to a receptor or
enzyme of interest. For example, the targeting peptide Q may be
LHRH, insulin, oxytosin, somatostatin, NK-1, VIP, GRP, bombesin or
any other hormone peptides known in the art, as well as analogues
and derivatives thereof.
In an alternative embodiment, a diagnostic or therapeutic agent is
attached to a diagnostic or therapeutic agent by a linker or specer
group of the present invention. Compounds of this embodiment may
generally have the formula: M-N-O-P-M
wherein M is a diagnostic or therapeutic agent, as defined above,
and N-O-P is the linker as defined above.
There is also provided a novel method of imaging using the
compounds of the present invention.
There is further provided a novel method for preparing a diagnostic
imaging agent comprising the step of adding to an injectable
imaging medium a substance containing the compounds of the present
invention.
A single or multi-vial kit that contains all of the componenets
needed to prepare the diagnostic or therapeutic agents of the
invention is an integral part of the present invention.
Novel methods of therapy (including radiotherapy and phototherapy)
using the compounds of the invention is also provided, as is a
novel method for preparing a radiotherapeutic agent comprising the
step of adding to an injectable therapeutic medium a substance
comprising a compound of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a graphical representation of the synthesis of
4-[[(3.beta.,5.beta.,
12.alpha.)-23-carboxy-12-hydroxy-24-norcholan-3-yl]amino]-4-oxobutanoic
acid N-hydroxysuccinimidyl ester (Compound A-OSu) as described in
Example I;
FIG. 2 is a chemical structure of Compound B as described in
Example II;
FIG. 3 is a graphical representation of a series of chemical
reactions for the synthesis of
(3.beta.,5.beta.,12.alpha.)-3-[[3,5-Bis[[4-[(2,5-dioxo-1-pyrrolidinyl)oxy-
]-1,4-dioxobutyl]amino]benzoyl]amino]-12-hydroxycholan-24-oic acid
1,1-dimethylethyl ester (Compound C--(OSu).sub.2), as described in
Example III;
FIG. 4 is a chemical structure of bovine
1-[[(3.beta.,5.beta.,12.alpha.)-23-[(1,1-dimethyl)ethoxycarbonyl]-12-hydr-
oxy-24-norcholan-3-yl]amino]carbonyl-3,5-bis[[4-(insulin-N.sup.eB29-yl)-1,-
4-dioxobutyl]amino]benzene (Compound D), as described in Example
III;
FIG. 5A is a chemical structure of
N.sup.1,N.sup.4,N.sup.7-triacetato-1,4,7,10-tetraazacyclododecan-N.sup.10-
-(2-acetamido ethylamine) (Compound E), as described in Example
VII;
FIG. 5B is a chemical structure of Compound F, as described in
Example VII;
FIG. 6A is a graphical representation of a series of chemical
reactions for the synthesis of intermediate C ((3.beta.,
5.beta.)-3-(9H-Fluoren-9-ylmethoxy)aminocholan-24-oic acid), from A
(Methyl-(3.beta., 5.beta.)-3-aminocholan-24-ate) and B ((3.beta.,
5.beta.)-3-aminocholan-24-oic acid), as described in Example
VIII;
FIG. 6B is a graphical representation of the sequential reaction
for the synthesis of
N-[(3.beta.,5.beta.)-3-[[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazac-
yclododec-1-yl]acetyl]amino]acetyl]amino]cholan-24-yl]-L-glutaminyl-L-tryp-
tophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide
(L62), as described in Example VIII;
FIG. 7A is a graphical representation of a series of chemical
reactions for the synthesis of intermediate
(3.beta.,5.beta.)-3-[[(9H-Fluoren-9-ylmethoxy)amino]acetyl]amino-12-oxoch-
olan-24-oic acid (C), as described in Example IX;
FIG. 7B is a graphical representation of the sequential reaction
for the synthesis of
N-[(3.beta.,5.beta.)-3-[[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazac-
yclododec-1-yl]acetyl]amino]acetyl]amino]-12,24-dioxocholan-24-yl]-L-gluta-
minyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methionin-
amide (L67), as described in Example
FIG. 7C is a chemical structure of
(3.beta.,5.beta.)-3-Amino-12-oxocholan-24-oic acid (B), as
described in Example IX;
FIG. 7D is a chemical structure of
(3.beta.,5.beta.)-3-[[(9H-Fluoren-9-ylmethoxy)amino]acetyl]amino-12-oxoch-
olan-24-oic acid (C), as described in Example IX;
FIG. 7E is a chemical structure of
N-[(3.beta.,5.beta.)-3-[[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazac-
yclododec-1-yl]acetyl]amino]acetyl]amino]-12,24-dioxocholan-24-yl]-L-gluta-
minyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methionin-
amide (L67), as described in Example IX;
FIG. 8A is a graphical representation of a sequence of reactions to
obtain intermediates (3.beta.,5.beta.,
12.alpha.)-3-[[(9H-Fluoren-9-ylmethoxy)amino]acetyl]amino-12-hydroxy
cholan-24-oic acid (3a) and
(3.beta.,5.beta.,7.alpha.,12.alpha.)-3-[[(9H-Fluoren-9ylmethoxy)amino]ace-
tyl]amino-7,12-dihydroxycholan-2.sup.4-oic acid (3b), as described
in Example X;
FIG. 8B is a graphical representation of the sequential reaction
for the synthesis of
N-[(3.beta.,5.beta.,12.alpha.)-3-[[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-
-tetraazacyclododec-1-yl]acetyl]amino]acetyl]amino]-12-hydroxy-24-oxochola-
n-24-yl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-le-
ucyl-L-methioninamide (L63);
N-[(3.beta.,5.beta.,7.alpha.,12.alpha.)-3-[[[[[4,7,10-Tris(carboxymethyl)-
-1,4,7,10-tetraazacyclo
dodec-1-yl]acetyl]amino]acetyl]amino]-7,12-dihydroxy-24-oxocholan-24-yl]--
L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-me-
thioninamide (L64); and
N-[(3.beta.,5.beta.)-3-[[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazac-
yclododec-1-yl]acetyl]amino]
acetyl]amino]-12,24-dioxocholan-24-yl]-L-glutaminyl-L-tryptophyl-L-alanyl-
-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide (L67) as
described in Example X;
FIG. 8C is a graphical representation of the sequential reaction
for the synthesis of
N-[(3.beta.,5.beta.,7.alpha.,12.alpha.)-3-[[[[[4,7,10-Tris(carboxymethyl)-
-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]acetyl]amino]-7,12-dihydrox-
y-24-oxocholan-24-yl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L--
histidyl-L-leucyl-L-methioninamide (L64), as described in Example
X;
FIG. 8D is a chemical structure of
(3.beta.,5.beta.,7.alpha.,12.alpha.)-3-amino-7,12-dihydroxycholan-24-oic
acid (2b), as described in Example X;
FIG. 8E is a chemical structure of
(3.beta.,5.beta.,12.alpha.)-3-[[(9H-Fluoren-9-ylmethoxy)amino]acetyl]amin-
o-12-hydroxycholan-24-oic acid (3a), as described in Example X;
FIG. 8F is a chemical structure of
(3.beta.,5.beta.,7.alpha.,12.alpha.)-3-[[(9H-Fluoren-9-ylmethoxy)amino]ac-
etyl]amino-7,12-dihydroxycholan-24-oic acid (3b), as described in
Example X;
FIG. 8G is a chemical structure of
N-[(3.beta.,5.beta.,12.alpha.)-3-[[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-
-tetraazacyclododec-1-yl]acetyl]amino]acetyl]amino]-12-hydroxy-24-oxochola-
n-24-yl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-le-
ucyl-L-methioninamide (L63), as described in Example X;
FIG. 8H is a chemical structure of
N-[(3.beta.,5.beta.,7.alpha.,12.alpha.)-3-[[[[[4,7,10-Tris(carboxymethyl)-
-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]acetyl]amino]-7,12dihydroxy-
-24-oxocholan-24-yl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-h-
istidyl-L-leucyl-L-methioninamide (L64), as described in Example
X;
FIG. 9 is a chemical structure of
DO3A-monoamide-Gly-Lys-(3,6,9-trioxaundecane-1,11-dicarboxylic
acid-3,7-dideoxy-3-aminocholic
acid)-L-arginyl-L-glutaminyl-L-triptophyl-L-alanyl-L-valyl-glycyl-L-histi-
dyl-L-leucyl-L-methioninamide (L65);
FIG. 10A is a chemical structure of
N-[2-S-[[[[[12.alpha.-Hydroxy-17.beta.-(1-methyl-3-carboxypropyl)etiochol-
an-3
B-carbamoylmnethoxyethoxyethoxyacetyl]-amino-6-[4,7,10-tris(carboxyme-
thyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]acetyl]amino]hexanoyl--
L-glutaminyl-L-trtophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-meth-
ioninamide (L66);
FIG. 10B is a chemical structure
N-[(3.beta.,5.beta.,7.alpha.,12.alpha.)-3-[[[[[[4,7,10-Tris(carboxymethyl-
)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]ethoxyethoxy]acetyl]amino]-
-7,12-dihydroxycholan-24-yl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-gl-
ycyl-L-histidyl-L-leucyl-L-methioninamide (L69);
FIG. 11A is a graphical representation of the results of HSA
binding experiments with Gd-L64 described in Example XV;
FIG. 11B is a graphical representation of the results of HSA
binding experiments with Gd-L64 described in Example XV;
FIG. 12A is a graphical representation of the results of
radiotherapy experiments described in Example XVII; and
FIG. 12B is a graphical representation of the results of other
radiotherapy experiments described in Example XVII.
FIG. 13A is a graphical representation of a reaction for the
synthesis of
(3.beta.,5.beta.,7.alpha.,12.alpha.)-3-(9H-Fluoren-9-ylmethoxy)amino-7,12-
-dihydroxycholan-24-oic acid (B) as described in Example XX.
FIG. 13B is a graphical representation of a reaction for the
synthesis of
N-[(3.beta.,5.beta.,7.alpha.,12.alpha.)-3-[[[2-[2-[[[4,7,10-Tris(carboxym-
ethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]ethoxy]ethoxy]acetyl]-
amino]-7,12-dihydroxy-24-oxocholan-24-yl]-L-glutaminyl-L-tryptophyl-L-alan-
yl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide, (L69), as
described in Example XX.
DETAILED DESCRIPTION OF THE INVENTION
In the following description, various aspects of the present
invention will be further elaborated. For purposes of explanation,
specific configurations and details are set forth in order to
provide a thorough understanding of the present invention. However,
it will also be apparent to one skilled in the art that the present
invention may be practiced without the specific details.
Furthermore, well known features may be omitted or simplified in
order not to obscure the present invention.
In an embodiment of the present invention, there is provided a new
and improved method for improving the half life of a pharmaceutical
compound for use in diagnosis or therapy, as well as compounds
exhibiting such improved half life. In these embodiments, a
diagnostic or therapeutic agent (such as a metal chelator, a
radioactive halogen, or an optical label) is attached to a
targeting peptide by a linker or spacer group of the present
invention.
In general, compounds of the invention comprise compounds of the
formula: M-N-O-P-Q wherein M is the diagnostic or therapeutic
agent, N-O-P is the linker, and Q is the targeting moiety. As
explained in more detail infra, M and Q may be attached at either
end of the linker (e.g. at an amino or a carboxylic acid group of a
cholic acid derivative of the invention) or M and Q may both be
attached to the same end of the linker (e.g. both to an amino or
both to a carboxylic acid group of a cholic acid derivative linker
of the invention).
In an alternative embodiment, a diagnostic or therapeutic agent is
attached to a second diagnostic or therapeutic agent by a linker or
spacer group of the present invention. Compounds of this embodiment
may generally have the formula: M-N-O-P-M wherein M is a diagnostic
or therapeutic agent, as defined above, and N-O-P is the linker as
defined above. As explained in more detail infra, each M may be
attached at either end of the linker (e.g. at an amino or a
carboxtlic acid group of a cholic acid derivative linker of the
invention) or both M may be attached to the same end of the linker
(e.g. both attached to an amino or carboxylic acid groups of a
cholic acid derivative linker of the invention).
Each of the diagnostic or therapeutic agent, linker, and targeting
moiety is described in the discussion that follows. When M is a
metal chelator, it may be in the form complexed with a metal
radionuclide or not. Alternatively, M may be a radioactive halogen
instead of a metal chelator. In another preferred embodiment, M is
an optical label (e.g. a photolabel or other label detectable by
light imaging, optoacoustical imaging or photoluminescence or
useful in phototherapy).
In another embodiment of the present invention, there is provided a
new and improved linker or spacer group which is capable of
improving the half life of a pharmaceutical compound when used to
link a diagnostic or therapeutic agent to a targeting moiety or to
link a diagnostic or therapeutic agent to a second diagnostic or
therapeutic agent. In general, linkers of the present invention may
have the formula: N-O-P wherein each of N, O and P are defined
throughout the specification.
Compounds meeting these criteria have improved pharmacokinetic
properties compared to other radiolabeled targeting peptide
conjugates known in the art. For example, compounds prepared with
the linkers of the present invention appear to be retained in the
bloodstream longer (consistent with binding to human serum albumin
(HSA)), and thus will have a longer half life than prior known
compounds. The longer half life is medically beneficial because it
permits longer diagnostic imaging time, or for therapeutic uses,
longer exposure to of the targeted cells and tumors to radioactive
treatment.
1A. Metal Chelator
The term "metal chelator" refers to a molecule that forms a complex
with a metal atom, wherein said complex is stable under
physiological conditions. That is, the metal will remain complexed
to the chelator backbone in vivo. More particularly, a metal
chelator is a molecule that complexes to a radionuclide metal to
form a metal complex that is stable under physiological conditions
and which also has at least one reactive functional group for
conjugation with the linker N-O-P. The metal chelator M may be any
of the metal chelators known in the art for complexing a medically
useful metal ion or radionuclide. The metal chelator may or may not
be complexed with a metal radionuclide. Furthermore, the metal
chelator can include an optional spacer such as, for example, a
single amino acid (e.g., Gly) which does not complex with the
metal, but which creates a physical separation between the metal
chelator and the linker.
The metal chelators of the invention may include, for example,
linear, macrocyclic, terpyridine, and N.sub.3S, N.sub.2S.sub.2, or
N.sub.4 chelators (see also, U.S. Pat. Nos. 5,367,080, 5,364,613,
5,021,556, 5,075,099, 5,886,142, the disclosures of which are
incorporated by reference herein in their entirety), and other
chelators known in the art including, but not limited to, HYNIC,
DTPA, EDTA, DOTA, TETA, and bisamino bisthiol (BAT) chelators (see
also U.S. Pat. No. 5,720,934). For example, N.sub.4 chelators are
described in U.S. Pat. Nos. 6,143,274; 6,093,382; 5,608,110;
5,665,329; 5,656,254; and 5,688,487, the disclosures of which are
incorporated by reference herein in their entirety. Certain
N.sub.3S chelators are described in PCT/CA94/00395, PCT/CA94/00479,
PCT/CA95/00249 and in U.S. Pat. Nos. 5,662,885; 5,976,495; and
5,780,006, the disclosures of which are incorporated by reference
herein in their entirety. The chelator may also include derivatives
of the chelating ligand mercapto-acetyl-glycyl-glycyl-glycine
(MAG3), which contains an N.sub.3S, and N.sub.2S.sub.2 systems such
as MAMA (monoamidemonoaminedithiols), DADS (N.sub.2S
diaminedithiols), CODADS and the like. These ligand systems and a
variety of others are described in Liu and Edwards, Chem Rev. 1999,
99, 2235-2268 and references therein, the disclosures of which are
incorporated by reference herein in their entirety.
The metal chelator may also include complexes containing ligand
atoms that are not donated to the metal in a tetradentate array.
These include the boronic acid adducts of technetium and rhenium
dioximes, such as those described in U.S. Pat. Nos. 5,183,653;
5,387,409; and 5,118,797, the disclosures of which are incorporated
by reference herein, in their entirety.
Examples of preferred chelators include, but are not limited to,
diethylenetriamine pentaacetic acid (DTPA),
1,4,7,10-tetraazacyclotetradecane-1,4,7,10-tetraacetic acid (DOTA),
1-substituted 1,4,7,-tricarboxymethyl 1,4,7,10
tetraazacyclododecane triacetic acid (DO3A),
ethylenediaminetetraacetic acid (EDTA), and
1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA).
Additional chelating ligands are
ethylenebis-(2-hydroxy-phenylglycine) (EHPG), and derivatives
thereof, including 5-Cl-EHPG, 5-Br-EBPG, 5-Me-EHPG, 5-t-Bu-EHPG,
and 5-sec-Bu-EHPG; benzodiethylenetriamine pentaacetic acid
(benzo-DTPA) and derivatives thereof, including dibenzo-DTPA,
phenyl-DTPA, diphenyl-DTPA, benzyl-DTPA, and dibenzyl-DTPA; bis-2
(hydroxybenzyl)-ethylene-diaminediacetic acid (HBED) and
derivatives thereof; the class of macrocyclic compounds which
contain at least 3 carbon atoms, more preferably at least 6, and at
least two heteroatoms (O and/or N), which macrocyclic compounds can
consist of one ring, or two or three rings joined together at the
hetero ring elements, e.g., benzo-DOTA, dibenzo-DOTA, and
benzo-NOTA, where NOTA is 1,4,7-triazacyclononane
N,N',N''-triacetic acid, benzo-TETA, benzo-DOTMA, where DOTMA is
1,4,7,10-tetraazacyclotetradecane-1,4,7,10-tetra(methyl tetraacetic
acid), and benzo-TETMA, where TETMA is
1,4,8,11-tetraazacyclotetradecane-1,4,8,11-(methyl tetraacetic
acid); derivatives of 1,3-propylenediaminetetraacetic acid (PDTA)
and triethylenetetraaminehexaacetic acid (TTHA); derivatives of
1,5,10-N,N',N''-tris(2,3-dihydroxybenzoyl)-tricatecholate (LICAM)
and 1,3,5-N,N',N''-tris(2,3-dihydroxybenzoyl) aminomethylbenzene
(MECAM). Examples of representative chelators and chelating groups
contemplated by the present invention are described in WO 98/18496,
WO 86/06605, WO 91/03200, WO 95/28179, WO 96/23526, WO 97/36619,
PCT/US98/01473, PCT/US98/20182, and U.S. Pat. Nos. 4,899,755,
5,474,756, U.S 5,846,519, 6,143,274, co-pending application U.S.
Provisional Ser. No. 60/532,842, each of which is hereby
incorporated by reference in its entirety.
Particularly preferred metal chelators include those of Formula 1,
2 and 3 (for .sup.111In and radioactive lanthanides, such as, for
example .sup.177Lu, .sup.90Y, .sup.153Sm, and .sup.166Ho) and those
of Formula 4, 5 and 6 (for radioactive .sup.99mTc, .sup.186Re, and
.sup.188Re) set forth below. These and other metal chelating groups
are described in U.S. Pat. Nos. 6,093,382 and 5,608,110, which are
incorporated by reference herein in their entirety. Additionally,
the chelating group of Formula 3 is described in, for example, U.S.
Pat. No. 6,143,274; the chelating group of Formula 5 is described
in, for example, U.S. Pat. Nos. 5,627,286 and 6,093,382, and the
chelating group of Formula 6 is described in, for example, U.S.
Pat. Nos. 5,662,885; 5,780,006; and 5,976,495. Specific metal
chelators of Formula 6 include N,N-dimethyl-Gly-Ser-Cys;
N,N-dimethyl-Gly-Thr-Cys; N,N-diethyl-Gly-Ser-Cys;
N,N-dibenzyl-Gly-Ser-Cys; and other variations thereof. For
example, spacers which do not actually complex with the metal
radionuclide such as an extra single amino acid Gly, may be
attached to these metal chelators (e.g.,
N,N-dimethyl-Gly-Ser-Cys-Gly; N,N-dimethyl-Gly-Thr-Cys-Gly;
N,N-diethyl-Gly-Ser-Cys-Gly; N,N-dibenzyl-Gly-Ser-Cys-Gly). Other
useful metal chelators such as all of those disclosed in U.S. Pat.
No. 6,334,996, also incorporated by reference (e.g.,
Dimethyl-Gly-L-t-Butyl-Gly-L-Cys-Gly;
Dimethyl-Gly-D-t-Butyl-Gly-L-Cys-Gly;
Dimethyl-Gly-L-t-Butyl-Gly-L-Cys, etc.)
Furthermore, sulfur protecting groups such as Acm
(acetamidomethyl), trityl or other known alkyl, aryl, acyl,
alkanoyl, aryloyl, mercaptoacyl and organothiol groups may be
attached to the cysteine amino acid of these metal chelators.
Additionally, other useful metal chelators include:
##STR00001## ##STR00002##
In the above Formulas 1 and 2, R is alkyl, preferably methyl. In
the above Formulas 5a and 5b, X is either CH.sub.2 or O; Y is
C.sub.1-C.sub.10 branched or unbranched alkyl; aryl, aryloxy,
arylamino, arylaminoacyl; arylalkyl--where the alkyl group or
groups attached to the aryl group are C.sub.1-C.sub.10 branched or
unbranched alkyl groups, C.sub.1-C.sub.10 branched or unbranched
hydroxy or polyhydroxyalkyl groups or polyalkoxyalkyl or
polyhydroxy-polyalkoxyalkyl groups, J is optional, but if present
is C(.dbd.O)--, OC(.dbd.O)--, SO.sub.2--, NC(.dbd.O)--,
NC(.dbd.S)--, N(Y), NC(.dbd.NCH.sub.3)--, NC(.dbd.NH)--, N.dbd.N--,
homopolyamides or heteropolyamines derived from synthetic or
naturally occurring amino acids; all where n is 1-100. Other
variants of these structures are described, for example, in U.S.
Pat. No. 6,093,382. In Formula 6, the group S--NHCOCH.sub.3 may be
replaced with SH or S-Z wherein Z is any of the known sulfur
protecting groups such as those described above. Formula 7
illustrates one embodiment of t-butyl compounds useful as a metal
chelator. The disclosures of each of the foregoing patents,
applications and references are incorporated by reference herein,
in their entirety.
In a preferred embodiment, the metal chelator includes cyclic or
acyclic polyaminocarboxylic acids such as DOTA
(1,4,7,10-tetraazacyclododecane-1,4,7,10tetraacetic acid), DTPA
(diethylenetriaminepentaacetic acid), DTPA-bismethylamide,
DTPA-bismorpholineamide, DO3A
N-[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl,
HP-DO3A, DO3A-monoamide and derivatives thereof.
Preferred metal radionuclides for scintigraphy or radiotherapy
include .sup.99mTc, .sup.51Cr, .sup.67Ga, .sup.68Ga, .sup.47SC,
.sup.51Cr, .sup.167Tm, .sup.141Ce, .sup.111In, .sup.168Yb,
.sup.175Yb, .sup.140La, .sup.90Y, .sup.88Y, .sup.153Sm, .sup.166Ho,
.sup.165Dy, .sup.166Dy, .sup.62Cu, .sup.64Cu, .sup.67Cu, .sup.97Ru,
.sup.103Ru, .sup.186Re, .sup.188Re, .sup.203Pb, .sup.211Bi,
.sup.212Bi, .sup.213Bi, .sup.214Bi, .sup.105Rh, .sup.109Pd,
.sup.117mSn, .sup.149Pm, .sup.161Th, .sup.177Lu, .sup.198Au and
.sup.199Au and oxides or nitrides thereof. The choice of metal will
be determined based on the desired therapeutic or diagnostic
application. For example, for diagnostic purposes (e.g., to
diagnose and monitor therapeutic progress in primary tumors and
metastases), the preferred radionuclides include .sup.64Cu,
.sup.67Ga, .sup.68Ga, .sup.99mTc, and .sup.111In, with .sup.99mTc
and .sup.111In being especially preferred. For therapeutic purposes
(e.g., to provide radiotherapy for primary tumors and metastasis
related to cancers of the prostate, breast, lung, etc.), the
preferred radionuclides include .sup.64Cu, .sup.90Y, .sup.105Rh,
.sup.111In, .sup.117mSn, .sup.149Pm, .sup.153Sm, .sup.161Tb,
.sup.166Dy, .sup.166Ho, .sup.175Yb, .sup.177Lu, .sup.186Re,
.sup.188Re, and .sup.199Au, with .sup.177Lu, .sup.90Y, .sup.186Re
and .sup.188Re being particularly preferred. .sup.99mTc is
particularly useful and is a preferred diagnostic radionuclide
because of its low cost, availability, imaging properties, and high
specific activity. The nuclear and radioactive properties of
.sup.99mTc make this isotope an ideal scintigraphic imaging agent.
This isotope has a single photon energy of 140 keV and a
radioactive half-life of about 6 hours, and is readily available
from a .sup.99Mo-.sup.99mTc generator. For example, the .sup.99mTc
labeled peptide can be used to diagnose and monitor therapeutic
progress in primary tumors and metastases. Peptides labeled with
.sup.177Lu, .sup.90Y or other therapeutic radionuclides can be used
to provide radiotherapy for primary tumors and metastases related
to cancers of the prostate, breast, lung, etc.
1B. Radioactive Halogens
In an alternative embodiment, the compounds of the present
invention can be labeled by halogenation using radionuclides, such
as .sup.18F, .sup.124I, .sup.125I, .sup.131I, .sup.123I, .sup.77Br,
and .sup.76Br instead of using a metal chelator to attach a metal
radionuclide. The choice of metal or halogen radionuclides will be
determined based on the desired therapeutic or diagnostic
application.
2A. Other Diagnostic Moieties
In alternative embodiments, compounds of the present invention can
incorporate other diagnostic moieties, such as agents which enable
detection of the compounds by such techniques as x-ray, magnetic
resonance imaging, ultrasound, fluorescence and other optical
imaging methodologies, as described in more detail below. The
choice of diagnostic moiety will be determined based on the desired
application. In one embodiment, the compounds of the invention may
be conjugated with photolabels, such as optical dyes, including
organic chromophores or fluorophores, having extensive delocalized
ring systems and having absorption or emission maxima in the range
of 400-1500 nm. The compounds of the invention may alternatively be
derivatized with a bioluminescent molecule. The preferred range of
absorption maxima for photolabels is between 600 and 1000 nm to
minimize interference with the signal from hemoglobin. Preferably,
photoabsorption labels have large molar absorptivities,
e.g.>10.sup.5 cm.sup.-1M.sup.-1, while fluorescent optical dyes
will have high quantum yields. Examples of optical dyes include,
but are not limited to those described in WO 98/18497, WO 98/18496,
WO 98/18495, WO 98/18498, WO 98/53857, WO 96/17628, WO 97/18841, WO
96/23524, WO 98/47538, and references cited therein. For example,
the photolabels may be covalently linked directly to compounds of
the invention, such as, for example, compounds comprised of
targeting peptides or diagnostic or therapeutic moieties and
linkers of the invention. Several dyes that absorb and emit light
in the visible and near-infrared region of electromagnetic spectrum
are currently being used for various biomedical applications due to
their biocompatibility, high molar absorptivity, and/or high
fluorescence quantum yields. The high sensitivity of the optical
modality in conjunction with dyes as contrast agents parallels that
of nuclear medicine, and permits visualization of organs and
tissues without the undesirable effect of ionizing radiation.
Cyanine dyes with intense absorption and emission in the
near-infrared (NIR) region are particularly useful, because
biological tissues are optically transparent in this region. For
example, indocyanine green, which absorbs and emits in the NIR
region has been used for monitoring cardiac output, hepatic
functions, and liver blood flow and its functionalized derivatives
have been used to conjugate biomolecules for diagnostic purposes
(R. B. Mujumdar, L. A. Ernst, S. R. Mujumdar, et al., Cyanine dye
labeling reagents: Sulfoindocyanine succinimidyl esters.
Bioconjugate Chemistry, 1993, 4(2), 105-111; Linda G. Lee and Sam
L. Woo. "N-Heteroaromatic ion and iminiun ion substituted cyanine
dyes for use as fluorescent labels", U.S. Pat. No. 5,453,505; Eric
Hohenschuh, et al. "Light imaging contrast agents", WO 98/48846;
Jonathan Turner, et al. "Optical diagnostic agents for the
diagnosis of neurodegenerative diseases by means of near infra-red
radiation", WO 98/22146; Kai Licha, et al. "In-vivo diagnostic
process by near infrared radiation", WO 96/17628; Robert A. Snow,
et al., Compounds, WO 98/48838. Various imaging techniques and
reagents are described in U.S. Pat. Nos. 6,663,847, 6,656,451,
6,641,798, 6,485,704, 6,423,547, 6,395,257, 6,280,703, 6,277,841,
6,264,920, 6,264,919, 6,228,344, 6,217,848, 6,190,641, 6,183,726,
6,180,087, 6,180,086, 6,180,085, 6,013,243, and published U.S.
Patent Applications 2003185756, 20031656432, 2003158127,
2003152577, 2003143159, 2003105300, 2003105299, 2003072763,
2003036538, 2003031627, 2003017164, 2002169107, 2002164287, and
2002156117.
2B. Other Therapeutic Moieties
In alternative embodiments, compounds of the present invention can
incorporate other therapeutic moieties such as antibiotics,
hormones, enzymes, antibodies, growth factors, as described in more
detail below. Alternatively, compounds of the invention may be
administered in combination with a therapeutic moiety. The choice
of therapeutic moiety will be determined based on the desired
application. Suitable therapeutic moieties include, but are not
limited to: antineoplastic agents, such as platinum compounds
(e.g., spiroplatin, cisplatin, and carboplatin), methotrexate,
adriamycin, mitomycin, ansamitocin, bleomycin, cytosine,
arabinoside, arabinosyl adenine, mercaptopolylysine, vincristine,
busulfan, chlorambucil, melphalan (e.g., PAM, a, L-PAM or
phennylalanine mustard), mercaptopurine, mitotane. procarbazine
hydrochloride, dactinomycin (actinomycin D), daunorubcin
hydrochloride, doxorubicin hydrochloride, taxol, mitomycin,
plicamycin (mithramycin), aminoglutethimide, estramustine phosphate
sodium, flutamide, leuprolide acetate, megestrol acetate, tamoxifen
citrate, testolactone, trilostane, amsacrine (m-AMSA), asparaginase
(L-asparaginase) Erwina aparaginase, etoposide (VP-16), interferon
.alpha.-2a, interferon .alpha.-2b, teniposide (VM-26), vinblastine
sulfate (VLB), and arabinosyl; blood products such as parenteral
iron, hemin, hematoporphyrins and their derivatives; biological
response modifiers such as muramyldipeptide, muramyltripeptide;
microbial cell wall components; lymphokines (e.g., bacterial
endotoxin such as lipopolysaccharide, macrophage activation
factor); sub-units of bacteria (such as Mycobacteria,
Corynebacteria); the synthetic dipeptide
N-acetyl-muramyl-1-alanyl-I)-isoglutamine; anti-fungal agents such
as ketoconazole, nystatin, griseofulvin, flucytosine (5-fc),
miconazole, amphotericin B, ricin, and .beta.-lactam antibiotics
(e.g., sulfazecin); hormones such as growth hormone, melanocyte
stimulating hormone, estradiol, beclomethasone, dipropionate,
betamethasone, betamethasone acetate and betamethasone sodium
phosphate, vetamethsone disodium phosphate, vetemthsone sodium
phosphate, cortisoneacetate, dexamethasone, dexamethasone acetate,
dexamethasone sodium phosphate, flunisolide, hydrocortisone,
hydrocortisone acetate, hydrocortisone cupionate, hydrocortisone
sodium phosphate, hydrocortisone sodium succinate,
methylprednisolone, methylprednisolone acetate, methylprednisolone
sodium succinate, paramethasone acetate, prednisolone, prednisotone
acetate, prednisolone sodium phosphate, prednisolone tebutate,
prednisone, triamcinolone, triamcinolone acetonide, triamcinolone
diacetate, triameinolone hexacetonide and fludrocortisone acetate;
vitamins such as cyanocobalamin neinoic acid, retinoids and
derivatives such as retinol palmitate, and .alpha.-tocpherol;
enzymes such as manganese super oxide dismutase or alkaline
phosphatase; anti-allergice agents such as amelexanox;
anti-coagulation agents such as phenprocoumon and heparin;
circulatory drugs such as propranolol; metabolic potentiators such
as glutathione; antituberculars such as para-aminosalicylic acid,
isoniazid, capreomycin sulfate cycloscrine, ethambutol
hydrochloride ethionamide, pyrazinamide, rifampin, and streptomycin
sulfate; antivirals such as acyclovir, amantadine azidothymidine
(AZT or Zidovudine), ribavirin and vidarabine monohydrate (adenine
arahinoside, ara-A); antianginals such as diltiazem, nifedipine,
verapamil, erythritol tetranitrate, isosorbide dinitrate,
nitroglycerin (glyceryl trinitrate) and pentaerythritol
tetranitrate; antibiotics, anti-inflammatories such as diflunisal,
ibuprofen, indomethacin, meclofenamate, mefenamic acid, naproxen,
oxyphenbutazone, phenylbutazone, piroxicam, sulindac, tolmetin,
aspirin and salicylates; antiprotozoans such as chloroquine,
hydroxychloroquine, metroidazole, quinine and meglumine antimonate;
antirheumatics such as penicillamine; narcotics such as paregoric;
opiates such as codeine, heroin, methadone, morphine and opium;
cardiac glycosides such as deslanoside, digitoxin, digoxin,
digitalin and digitalis; neuromuscular blockers such as atracutrium
mesylate, gallamice triethiodide, hexafluorenium bromide,
metocurine iodide, pancuronium bromide, succinylcholine chloride
(suxamethonium chloride), tubocurarine chloride and vecuronium
bromide; sedatives (hypnotics) such as amobarbital, amobarbital
sodium, aprobarbital, butabarbital sodium, chloral hydrate,
ethchlorvynol, ethinamate, flurazepam hydrochloride, glutethimide,
methotrimeprazine hydrochloride, methyprylon, midazolam
hydrochloride, paraldehyde, pentobarbital, pentobarbital sodium,
phenobarbital sodium, secobarbital sodium, talbutal, temazepam and
triazolam; local anesthetics such as bupivacaine hydrochloride,
chloroprocaine hydrochloride, etidocaine hydrochloride, lidocaine
hydrochloride, mepivacaine hydrochloride, procaine hydrochloride
and tetracaine hydrochloride; and general anesthetics such as
droperidol, etomidate, fentanyl citrate with droperidol, ketamine
hydrochloride, methohexital sodium and thiopental sodium. In
certain embodiments, the therapeutic may be monoclonal antibody,
such as a monoclonal antibody capable of binding to melanoma
antigen.
3. Linkers Containing at Least One Substituted Bile Acid
In an exemplary embodiment of the present invention, the linker
N-O-P contains at least one substituted bile acid. Thus, in this
embodiment of the linker N-O-P,
N is 0 (where 0 means it is absent), an alpha amino acid, a
substituted bile acid or other linking group;
O is an alpha amino acid or a substituted bile acid; and
P is 0, an alpha amino acid, a substituted bile acid or other
linking group,
wherein at least one of N, O or P is a substituted bile acid.
Alpha amino acids are well known in the art and include naturally
occurring and synthetic amino acids.
Bile acids are found in bile (a secretion of the liver) and are
steroids having a hydroxyl group and a five carbon atom side chain
terminating in a carboxyl group. In substituted bile acids, at
least one atom such as a hydrogen atom of the bile acid is
substituted with another atom, molecule or chemical group. For
example, substituted bile acids include those having a 3-amino,
24-carboxyl function optionally substituted at positions 7 and 12
with hydrogen, hydroxyl or keto functionality.
Other useful substituted bile acids in the present invention
include substituted cholic acids and derivatives thereof. Specific
substituted cholic acid derivatives include:
(3.beta.,5.beta.)-3-aminocholan-24-oic acid;
(3.beta.,5.beta.,12.alpha.)-3-amino-12-hydroxycholan-24-oic acid;
(3.beta.,5.beta.,7.alpha.,12.alpha.)-3-amino-7,12-dihydroxycholan-24-oic
acid;
Lys-(3,6,9)-trioxaundecane-1,11-dicarbonyl-3,7-dideoxy-3-aminocholi-
c acid);
(3.beta.,5.beta.,7.alpha.)-3-amino-7-hydroxy-12-oxocholan-24-oic
acid; and (3.beta.,5.beta.,7.alpha.)-3-amino-7-hydroxycholan-24-oic
acid.
3A. Other Linking Groups
Other linking groups which may be used within the linker N-O-P
include a chemical group that serves to couple the diagnostic or
therapeutic moiety to the targeting peptide or the other diagnostic
or therapeutic moiety while not adversely affecting either the
targeting function of the targeting peptide or the diagnostic or
therapeutic function of the diagnostic or therapeutic moiety.
Suitable other linking groups include peptides (i.e., amino acids
linked together) alone, a non-peptide group (e.g., hydrocarbon
chain) or a combination of an amino acid sequence and a non-peptide
spacer.
In one embodiment, other linking groups for use within the linker
N-O-P include L-glutamine and hydrocarbon chains, or a combination
thereof.
In another embodiment, other linking groups for use within the
linker N-O-P include a pure peptide linking group consisting of a
series of amino acids (e.g.; diglycine, triglycine, gly-gly-glu,
gly-ser-gly, etc.).
In yet a further embodiment, other linking groups for use within
the linker N-O-P can also include a hydrocarbon chain [i.e.,
R.sub.1--(CH.sub.2).sub.n--R.sub.2] wherein n is 0-10, preferably
n=3 to 9, R.sub.1 is a group (e.g., H.sub.2N--, HS--, --COOH) that
can be used as a site for covalently linking the ligand backbone or
the preformed metal chelator or metal complexing backbone or other
diagnostic or therapeutic moiety; and R.sub.2 is a group that is
used for covalent coupling to the N-terminal NH.sub.2-group of a
given targeting peptide (e.g., R.sub.2 is an activated COOH group)
or other diagnostic or therapeutic moiety. Several chemical methods
for conjugating ligands (i.e., chelators) or chelates
(chelators/ligands complexed with a radionuclide) to biomolecules
(such as targeting peptides) have been well described in the
literature [Wilbur, 1992; Parker, 1990; Hermanson, 1996; Frizberg
et al., 1995]. One or more of these methods could be used to link
either the uncomplexed ligand (chelator) or the radiometal chelate
or other diagnostic or therapeutic moiety to the linker or to link
the linker to the targeting peptides or other diagnostic or
therapeutic moiety. These methods include the formation of acid
anhydrides, aldehydes, arylisothiocyanates, activated esters, or
N-hydroxysuccinimides [Wilbur, 1992; Parker, 1990; Hermanson, 1996;
Frizberg et al., 1995].
In a preferred embodiment, other linking groups for use within the
linker N-O-P may be formed from linker precursors having
electrophiles or nucleophiles as set forth below:
LP1: a linker precursor having on at least two locations of the
linker the same electrophile E1 or the same nucleophile Nu1;
LP2: a linker precursor having an electrophile E1 and on another
location of the linker a different electrophile E2;
LP3: a linker precursor having a nucleophile Nu1 and on another
location of the linker a different nucleophile Nu2; or
LP4: a linker precursor having one end functionalized with an
electrophile E1 and the other with a nucleophile Nu1.
The preferred nucleophiles Nu1/Nu2 include --OH, --NH, --NR, --SH,
--HN--NH.sub.2, --RN--NH.sub.2, and --RN--NHR', in which R' and R
are independently selected from the definitions for R given above,
but for R' is not H.
The preferred electrophiles E1/E2 include --COOH, --CH.dbd.O
(aldehyde), --CR.dbd.OR' (ketone), --RN--C.dbd.S, --RN--C.dbd.O,
--S-S-2-pyridyl, --SO.sub.2--Y, --CH.sub.2C(.dbd.O)Y, and
##STR00003##
wherein Y can be selected from the following groups:
##STR00004##
4. Targeting Moiety
The targeting moiety (i.e. Q in the formula M-N-O-P-Q) is any
molecule that has a binding affinity for particular site or a
specific metabolic function. The targeting moiety directs the
compounds of the invention to the appropriate site, or involves the
compounds in a reaction, where the desired diagnostic or
therapeutic activity will occur. In an exemplary embodiment, the
targeting moiety may be a peptide, equivalent, derivative or analog
thereof which functions as a ligand that binds to a particular
site. In another exemplary embodiment, the targeting moiety may be
an enzyme, or a molecule that binds to an enzyme. In another
exemplary embodiment, the targeting moiety may be an
antibiotic.
In a preferred embodiment, the targeting peptide is a peptide that
binds to a receptor or enzyme of interest. For example, the
targeting peptide Q may be a peptide hormone such as, for example,
luetinising hormone releasing hormone (LHRH) such as that described
in the literature [e.g., Radiometal-Binding Analogues of
Leutenizing Hormone Releasing Hormone PCT/US96/08695;
PCT/US97/12084 (WO 98/02192)]; insulin; oxytosin; somatostatin;
Neuro kinin-1 (NK-1); Vasoactive Intestinal Peptide (VIP) including
both linear and cyclic versions as delineated in the literature,
[e.g., Comparison of Cyclic and Linear Analogs of Vasoactive
Intestinal Peptide. D. R. Bolin, J. M. Cottrell, R. Garippa, N.
Rinaldi, R. Senda, B. Simkio, M. O'Donnell. Peptides: Chemistry,
Structure and Biology Pravin T. P. Kaumaya, and Roberts S. Hodges
(Eds). Mayflower Scientific LTD., 1996, pgs 174-175]; gastrin
releasing peptide (GRP); bombesin and other known hormone peptides,
as well as analogues and derivatives thereof.
Other useful targeting peptides include analogues of somatostatin
which, for example, are Lanreotide
(Nal-Cys-Thr-DTrp-Lys-Val-Cys-Thr-NH.sub.2), Octreotide
(Nal-Cys-Thr-DTrp-Lys-Val-Cys-Thr-ol), and Maltose
(Phe-Cys-Thr-DTrp-Lys-Val-Cys-Thr-ol). These analogues are
described in the literature [e.g., Potent Somatostatin Analogs
Containing N-terminal Modifications, S. H. Kim, J. Z. Dong, T. D.
Gordon, H. L. Kimball, S. C. Moreau, J.-P. Moreau, B. A. Morgan, W.
A. Murphy and J. E. Taylor; Peptides: Chemistry, Structure and
Biology Pravin T. P. Kaumaya, and Roberts S. Hodges (Eds),
Mayflower Scientific LTD., 1996, pgs 241-243.]
Still other useful targeting peptides include Substance P agonists
[e.g., G. Bitan, G. Byk, Y. Mahriki, M. Hanani, D. Halle, Z.
Selinger, C. Gilon, Peptides: Chemistry, Structure and Biology,
Pravin T. P. Kaumaya, and Roberts S. Hodges (Eds), Mayflower
Scientific LTD., 1996, pgs 697-698; G Protein Antagonists A novel
hydrophobic peptide competes with receptor for G protein binding,
Hidehito Mukai, Eisuke Munekata, Tsutomu Higashijima, J. Biol.
Chem. 1992, 267, 16237-16243]; NPY(Y1) [e.g., Novel Analogues of
Neuropeptide Y with a Preference for the Y1-receptor, Richard M.
Soll, Michaela, C. Dinger, Ingrid Lundell, Dan Larhammer, Annette
G. Beck-Sickinger, Eur. J. Biochem. 2001, 268, 2828-2837;
99mTc-Labeled Neuropeptide Y Analogues as Potential Tumor Imaging
Agents, Michael Langer, Roberto La Bella, Elisa Garcia-Garayoa,
Annette G. Beck-Sickinger, Bioconjugate Chem. 2001, 12, 1028-1034;
Novel Peptide Conjugates for Tumor-Specific Chemotherapy, Michael
Langer, Felix Kratz, Barbara Rothen-Rutishauser, Heidi
Wnderli-Allenspach, Annette G. Beck-Sickinger, J. Med. Chem. 2001,
44, 1341-1348]; oxytocin; endothelin A and endothelin B;
bradykinin; Epidural Growth Factor (EGF); Interleukin-1 [Anti-IL-1
Activity of Peptide Fragments of IL-1 Family Proteins, I. Z.
Siemion, A. Kluczyk, Zbigtniew Wieczorek, Peptides 1998, 19,
373-382]; and cholecystokinin (CCK-B) [Cholecystokinin Receptor
Imaging Using an Octapeptide DTPA-CCK Analogue in Patients with
Medullary Thryroid Carcinoma, Eur. J. Nucl Med. 200, 27,
1312-1317].
Literature which gives a general review of targeting peptides, can
be found, for example, in the following: The Role of Peptides and
Their Receptors as Tumor Markers, Jean-Claude Reubi,
Gastrointestinal Hormones in Medicine, Pg 899-939; Peptide
Radiopharmaceutical in Nuclear Medicine, D. Blok, R. I. J. Feitsma,
P. Vermeij, E. J. K. Pauwels, Eur. J. Nucl Med. 1999, 26,
1511-1519; and Radiolabeled Peptides and Other Ligands for
Receptors Overexpressed in Tumor Cells for Imaging Neoplasms, John
G. McAfee, Ronald D. Neumann, Nuclear Medicine and Biology, 1996,
23, 673-676 (somatostatin, VIP, CCK, GRP, Substance P, Galanan,
MSH, LHRH, Arginine-vasopressin, endothelin). All of the
aforementioned literature in the preceding paragraphs are herein
incorporated by reference in their entirety.
Other targeting peptide references include the following:
Co-expressed peptide receptors in breast cancer as a molecular
basis of in vivo multireceptor tumour targeting. Jean Claude Reubi,
Mathias Gugger, Beatrice Waser. Eur. J. Nucl Med. 2002, 29,
855-862, (includes NPY, GRP); Radiometal-Binding Analogues of
Leutenizing Hormone Releasing Hormone PCT/US96/08695 (LHRH);
PCT/US97/12084 (WO 98/02192) (LHRH); PCT/EP90/01169 (radiotherapy
of peptides); WO 91/01144 (radiotherapy of peptides); and
PCT/EP00/01553 (molecules for the treatment and diagnosis of
tumours), all of which are herein incorporated by reference in
their entirety.
Additionally, analogues of a targeting peptide can be used. These
analogues include molecules that target a desired site receptor
with avidity that is greater than or equal to the targeting peptide
itself, as well as muteins, retropeptides and
retro-inverso-peptides of the targeting peptide. One of ordinary
skill will appreciate that these analogues may also contain
modifications which include substitutions, and/or deletions and/or
additions of one or several amino acids, insofar that these
modifications do not negatively alter the biological activity of
the peptides described therein. These substitutions may be carried
out by replacing one or more amino acids by their synonymous amino
acids. Synonymous amino acids within a group are defined as amino
acids that have sufficiently similar physicochemical properties to
allow substitution between members of a group in order to preserve
the biological function of the molecule. Synonymous amino acids as
used herein include synthetic derivatives of these amino acids
(such as for example the D-forms of amino acids and other synthetic
derivatives).
Deletions or insertions of amino acids may also be introduced into
the defined sequences provided they do not alter the biological
functions of said sequences. Preferentially such insertions or
deletions should be limited to 1, 2, 3, 4 or 5 amino acids and
should not remove or physically disturb or displace amino acids
which are critical to the functional conformation. Muteins of the
peptides or polypeptides described herein may have a sequence
homologous to the sequence disclosed in the present specification
in which amino acid substitutions, deletions, or insertions are
present at one or more amino acid positions. Muteins may have a
biological activity that is at least 40%, preferably at least 50%,
more preferably 60-70%, most preferably 80-90% of the peptides
described herein. However, they may also have a biological activity
greater than the peptides specifically exemplified, and thus do not
necessarily have to be identical to the biological function of the
exemplified peptides. Analogues of targeting peptides also include
peptidomimetics or pseudopeptides incorporating changes to the
amide bonds of the peptide backbone, including thioamides,
methylene amines, and E-olefins. Also peptides based on the
structure of a targeting peptide or its peptide analogues with
amino acids replaced by N-substituted hydrazine carbonyl compounds
(also known as aza amino acids) are included in the term analogues
as used herein.
The targeting peptide may be attached to the linker via the N or C
terminus or via attachment to the epsilon nitrogen of lysine, the
gamma nitrogen or ornithine or the second carboxyl group of
aspartic or glutamic acid.
In an exemplary embodiment, the targeting peptide Q is LHRH or an
analogue or derivative thereof. For example, it is well known in
the art that position 6 of LHRH agonists may be substituted with
different functional groups, such as, for example DLysine. In a
preferred embodiment the targeting peptide Q is an LHRH analogue of
the formula PGlu-His-Trp-W-Tyr-DLys-X-Y-Pro-Z, wherein
W=Ser, NMeSer, or Thr.
X=Leu, NMeLeu, t-ButylGly.
Y=Arg, Arg(Et2), Cit, Lys(isopropyl).
Z=Gly-NH.sub.2, NHEthyl, Azagly-NH.sub.2.
Linkers of the invention coupled to glycine and DLysine can be
attached to the LHRH analogue at position 6. This embodiment
includes compounds of the formula:
PGlu-His-Trp-W-Tyr-DLys(M-N-O-P)-X-Y-Pro-Z, wherein
W=Ser, NMeSer, or Thr.
X=Leu, NMeLeu, t-ButylGly.
Y=Arg, Arg(Et2), Cit, Lys(isopropyl).
Z=Gly-NH.sub.2, NHEthyl, Azagly-NH.sub.2.
M is a metal chelator as defined herein, and
N-O-P is a linker of the invention
In a particular preferred embodiment, the invention includes
compounds of the formula: PGlu-His-Trp-W-Tyr-DLys(DOTA-Gly-Cholic
acid derivative)-X-Y-Pro-Z, wherein
W=Ser, NMeSer, or Thr.
X=Leu, NMeLeu, t-ButylGly.
Y=Arg, Arg(Et2), Cit, Lys(isopropyl).
Z=Gly-NH.sub.2, NHEthyl, Azagly-NH.sub.2.
Here, DOTA-Gly is M (the metal chelator and a Gly spacer), N-O-P is
a linker of the present invention (e.g., a cholic acid derivative),
and Q is an LHRH incorporating DLys6 and the other substitutions
listed above.
In a preferred embodiment, the W, X, Y or Z components in the above
formulas are W=Ser, X=Leu, Y=Arg and Z=Gly-NH.sub.2.
In another preferred embodiment, Q is a peptide which targets a
receptor in the GRP receptor family, such as an analogue or
derivative of GRP or bombesin. Such targeting peptides are
discussed in co-pending U.S. Ser. No. 10/341,577 filed Jan. 13,
2003, as well as in U.S. Pat. No. 6,200,546, US 2002/0054855,
US2003/0224998, and WO 02/87637 which are incorporated by reference
in their entirety.
Examples of compounds having the formula M-N-O-P-Q which contain
linkers with at least one substituted bile acid which are attached
to a targeting peptide such as BBN(7-14) are listed in Table 1.
These compounds may be prepared using the methods disclosed herein,
particularly in the Examples, as well as by similar methods known
to those skilled in the art.
TABLE-US-00001 TABLE 1 Compounds Containing Linkers With At Least
One Substituted Bile Acid HPLC HPLC Compound method.sup.1 RT.sup.2
MS.sup.3 IC50.sup.5 M N O P G* L62 20-80% B 3.79 1741.2 >50
DO3A- Gly (3.beta.,5.beta.)-3- none BBN(7-- 14) monoamide
aminocholan- 24-oic acid L63 20-80% B 3.47 1757.0 23 DO3A- Gly
(3.beta.,5.beta.,12.alpha.)-3- none - BBN(7-14) monoamide amino-12-
hydroxycholan- 24-oic acid L64 20-50% B 5.31 1773.7 8.5 DO3A- Gly
(3.beta.,5.beta.,7.alpha.,12.alpha.- )- none BBN(7-14) monoamide
3-amino- 7,12- dihydroxycholan- 24-oic acid L65 20-80% B 3.57
2246.2 >50 DO3A- Gly Lys-(3,6,9- Arg BBN(7-14) monoamide
trioxaundecane- 1,11- dicarbonyl- 3,7- dideoxy-3- aminocholic acid)
L66 20-80% 3.79 2245.8 >50
(3.beta.,5.beta.,7.alpha.,12.alpha.)-3- Lys(- DO3A- Arg BBN(7-14)
amino-7,12- monoamide- dihydroxycholan-24-oic Gly) acid-3,6,9-
trioxaundecane-1,11- dicarbonyl L67 20-80% 3.25 1756.9 4.5 DO3A-
Gly (3.beta.,5.beta.,7.alpha.,12.alpha.)-- none BBN(7-14) monoamide
3-amino-12- oxacholan-24- oic acid L69 20-80% 3.25 1861.27 8 DO3A-
1- (3.beta.,5.beta.,7.alpha.,12.alpha.)- n- one BBN(7-14) monoamide
amino- 3-amino- 3,6- 7,12- dioxaoctanoic dihydroxycholan- acid
24-oic acid *BBN(7-14) is [SEQ ID NO: 1] .sup.1HPLC method refers
to the 10 minute time for the HPLC gradient. .sup.2HPLC RT refers
to the retention time of the compound in the HPLC. .sup.3MS refers
to mass spectra where molecular weight is calculated from mass/unit
charge (m/e). .sup.4IC.sub.50 refers to the concentration of
compound to inhibit 50% binding of iodinated bombesin to a GRP
receptor on cells.
As explained in more detail infra, compounds containing linkers of
the invention and GRP-R targeting peptides demonstrated
unexpectedly superior pharmacokinetics and tumor uptake in an
animal model.
The targeting peptide can be prepared by various methods depending
upon the selected chelator. The peptide can generally be most
conveniently prepared by techniques generally established and known
in the art of peptide synthesis, such as the solid-phase peptide
synthesis (SPPS) approach. Solid-phase peptide synthesis (SPPS)
involves the stepwise addition of amino acid residues to a growing
peptide chain that is linked to an insoluble support or matrix,
such as polystyrene. The C-terminal residue of the peptide is first
anchored to a commercially available support with its amino group
protected with an N-protecting agent such as a t-butyloxycarbonyl
group (Boc) or a fluorenylmethoxycarbonyl (Fmoc) group. The amino
protecting group is removed with suitable deprotecting agents such
as TFA in the case of Boc or piperidine for Fmoc and the next amino
acid residue (in N-protected form) is added with a coupling agent
such as N,N'-dicyclohexylcarbodiimide (DCC), or
N,N'-diisopropylcarbodiimide or
2-(1H-benzotriazol-1-yl)-1,1,3,3-tetrarethyluronium
hexafluorophosphate (HBTU). Upon formation of a peptide bond, the
reagents are washed from the support After addition of the final
residue, the peptide is cleaved from the support with a suitable
reagent such as trifluoroacetic acid (TFA) or hydrogen fluoride
(HF).
The linker may then be coupled to form a conjugate by reacting the
free amino group of a selected residue of the targeting peptide
with an appropriate functional group of the linker. The entire
construct of chelator, linker and targeting moiety discussed above
may also be assembled on resin and then cleaved by agency of
suitable reagents such as trifluoroacetic acid or HF, as well.
5. Labeling And Administration Of Radiopharmaceutical Compounds
Incorporation of the metal within radiopharmaceutical conjugates of
the invention can be achieved by various methods commonly known in
the art of coordination chemistry. When the metal is .sup.99mTc, a
preferred radionuclide for diagnostic imaging, the following
general procedure can be used to form a technetium complex. A
peptide-chelator conjugate solution is formed by initially
dissolving the conjugate in water, dilute acid, or in an aqueous
solution of an alcohol such as ethanol. The solution is then
optionally degassed to remove dissolved oxygen. When an --SH group
is present in the peptide, a thiol protecting group such as Acm
(acetamidomethyl), trityl or other thiol protecting group may
optionally be used to protect the thiol from oxidation. The thiol
protecting group(s) are removed with a suitable reagent, for
example with sodium hydroxide, and are then neutralized with an
organic acid such as acetic acid (pH 6.0-6.5). Alternatively, the
thiol protecting group can be removed in situ during technetium
chelation. In the labeling step, sodium pertechnetate obtained from
a molybdenum generator is added to a solution of the conjugate with
a sufficient amount of a reducing agent, such as stannous chloride,
to reduce technetium and is either allowed to stand at room
temperature or is heated. The labeled conjugate can be separated
from the contaminants .sup.99mTcO.sub.4.sup.- and colloidal
.sup.99mTcO.sub.2 chromatographically, for example with a C-18 Sep
Pak cartridge [Millipore Corporation, Waters Chromatography
Division, 34 Maple Street, Milford, Mass. 01757] or by HPLC using
methods known to those skilled in the art.
In an alternative method, the labeling can be accomplished by a
transchelation reaction. In this method, the technetium source is a
solution of technetium complexed with labile ligands prior to
reaction with the selected chelator, thus facilitating ligand
exchange with the selected chelator. Examples of suitable ligands
for transchelation includes tartrate, citrate, gluconate, and
heptagluconate. It will be appreciated that the conjugate can be
labeled using the techniques described above, or alternatively, the
chelator itself may be labeled and subsequently coupled to the
peptide to form the conjugate; a process referred to as the
"prelabeled chelate" method. Re and Tc are both in row VIIB of the
Periodic Table and they are chemical congeners. Thus, for the most
part, the complexation chemistry of these two metals with ligand
frameworks that exhibit high in vitro and in vivo stabilities are
the same [Eckelman, 1995] and similar chelators and procedures can
be used to label with Re. Many .sup.99mTc or .sup.186/188Re
complexes, which are employed to form stable radiometal complexes
with peptides and proteins, chelate these metals in their +5
oxidation state [Lister-James et al., 1997]. This oxidation state
makes it possible to selectively place .sup.99mTc- or
.sup.186/188Re into ligand frameworks already conjugated to the
biomolecule, constructed from a variety of .sup.99mTc(V) and/or
.sup.186/188Re(V) weak chelates (e.g., .sup.99mTc-glucoheptonate,
citrate, gluconate, etc.) [Eckelman, 1995; Lister-James et al.,
1997; Pollak et-al., 1996].
Labelling of a targeting peptide or other peptide-linker conjugate
of the invention with radioactive halogens can be done using any
method known in the art. The Examples describe several methods
which may be used.
A conjugate labeled with a radionuclide metal, such as .sup.99mTc,
can be administered to a mammal, including human patients or
subjects, by intravenous, subcutaneous or intraperitoneal injection
in a pharmaceutically acceptable carrier and/or solution such as
salt solutions like isotonic saline. Radiolabeled scintigraphic
imaging agents provided by the present invention are provided
having a suitable amount of radioactivity. In forming .sup.99mTc
radioactive complexes, it is generally preferred to form
radioactive complexes in solutions containing radioactivity at
concentrations of from about 0.01 millicurie (mCi) to 100 mCi per
mL. Generally, the unit dose to be administered has a radioactivity
of about 0.01 mCi to about 100 mCi, preferably 1 mCi to 30 mCi. The
solution to be injected at unit dosage is from about 0.01 mL to
about 10 mL. The amount of labeled conjugate appropriate for
administration is dependent upon the distribution profile of the
chosen conjugate in the sense that a rapidly cleared conjugate may
need to be administered in higher doses than one that clears less
rapidly. In vivo distribution and localization can be tracked by
standard scintigraphic techniques at an appropriate time subsequent
to administration; typically between thirty minutes and 180 minutes
depending upon the rate of accumulation at the target site with
respect to the rate of clearance at non-target tissue. For example,
after injection of the diagnostic radionuclide-labeled compounds of
the invention into the patient, a gamma camera calibrated for the
gamma ray energy of the nuclide incorporated in the imaging agent
can be used to image areas of uptake of the agent and quantify the
amount of radioactivity present in the site. Imaging of the site in
vivo can take place in a few minutes. However, imaging can take
place, if desired, hours or even longer, after the radiolabeled
peptide is injected into a patient. In most instances, a sufficient
amount of the administered dose will accumulate in the area to be
imaged within about 0.1 hour to permit the taking of
scintiphotos.
The compounds of the present invention can be administered to a
patient alone or as part of a composition that contains other
components such as excipients, diluents, radical scavengers,
stabilizers, and carriers, all of which are well-known in the art.
The compounds can be administered to patients either intravenously
or intraperitoneally.
There are numerous advantages associated with the present
invention. The compounds made in accordance with the present
invention form stable, well-defined .sup.99mTc or .sup.186/188Re
labeled compounds. Similar compounds of the invention can also be
made by using appropriate chelator frameworks for the respective
radiometals, to form stable, well-defined products labeled with
.sup.153Sm, .sup.90Y, .sup.166Ho, .sup.105Rh, .sup.199Au,
.sup.149Pm, .sup.177LU, .sup.111In or other radiometal. The
radioactive material that does not reach (i.e., does not bind) the
cancer cells is preferentially excreted efficiently into the urine
with minimal retention of the radiometal in the kidneys.
6. Alternative Embodiments
As explained in more detail in the Examples, compounds containing
the novel linkers of the present invention exhibit increased
affinity for serum albumin, which decreases the rate of excretion
for the compounds. These compounds thus exhibit longer half lives
than compounds without linkers of the invention. Surprisingly, this
property is exhibited both by compounds in which a targeting
peptide is attached to an amino group of a cholic acid derivative
linker of the invention, and also by compounds in which the
targeting peptide is attached via a carboxylic acid group of the
cholic acid derivative linker.
In an exemplary embodiment of the invention, the targeting peptide
may be attached to a cholic acid derivative linker of the invention
via an amino group at the 3-position. A diagnostic or therapeutic
moiety is attached via a carboxylic acid group at the
24-position.
In another exemplary embodiment, the targeting peptide may be
attached to a carboxylic acid group at the 24-position of the
cholic acid derivative linker of the invention. A diagnostic or
therapeutic moiety may be attached via an amino group at the
3-position of the linker. For example, in L62 (FIG. 6B) and L69
(FIG. 10B), the diagnostic moiety (a metal chelator) is attached at
the 3-position and the targeting peptide (BBN[7-14] is attached at
the 24-position of the cholic acid derivative linker.
In another exemplary embodiment, a diagnostic or therapeutic moiety
may be attached to each of the amino and carboxyl groups of a
cholic acid derivative linker. In a preferred exemplary embodiment,
a compound has a therapeutic moiety (an insulin molecule) attached
to the 3-amino group and another therapeutic moiety (an insulin
molecule) attached to the 24-carboxyl group of a cholic acid
derivative linker. See e.g. Compound D. (FIG. 4). In another
preferred exemplary embodiment, a compound has a therapeutic moiety
(an insulin molecule) attached to the 3-amino group and a
diagnostic moiety (a metal chelator) attached to the 24-carboxyl
group of a cholic acid derivative linker (or vice versa). See, e.g.
Compound F (FIG. 5B).
The invention also includes an embodiment in which a targeting
peptide and a diagnostic or therapeutic moiety (or two diagnostic
and/or therapeutic moieties) may both be attached via an amino
group at the 3-position of the cholic acid derivative linker. For
example, in L65 (FIG. 9) and L66 (FIG. 10A) the diagnostic moiety
(a metal chelator) and the targeting peptide (BBN[7-14]) are both
attached at the 3-position of the cholic acid derivative linker of
the invention.
7A. Diagnostic and Therapeutic Uses
When labeled with diagnostic and/or therapeutic moieties (such as,
for example, diagnostically or therapeutically useful metals),
compounds of the present invention can be used to treat and/or
detect diseases such as cancers, including tumors, by procedures
established in the arts of diagnostic imaging, radiodiagnostics and
radiotherapeutics. [Bushbaum, 1995; Fischman et al., 1993;
Schubiger et al., 1996; Lowbertz et al., 1994; Krenning et al.,
1994].
Compounds of the invention, which, as explained in more detail in
the Examples, show higher uptake in tumors in vivo than compounds
without the novel linkers disclosed herein, exhibit an improved
ability to target the desired receptor-expressing tissue (e.g.
receptor expressing tumors). Thus, compounds of the invention are
better able to image or deliver radiotherapy to these desired
tissues. Indeed, as shown in the Examples, radiotherapy is more
effective (and survival time increased) using compounds of the
invention.
The diagnostic application of these compounds can be as a first
line diagnostic screen for the presence of targeted cells using
diagnostic imaging (such as, for example, scintigraphic, magnetic
resonance, ultrasound, optical, photoacoustic or sonoluminescent
imaging), as an agent for targeting selected tissue using hand-held
radiation detection instrumentation in the field of radioimmuno
guided surgery (RIGS), as a means to obtain dosimetry data prior to
administration of the matched pair radiotherapeutic compound, and
as a means to assess a targeted receptor population as a function
of treatment over time.
The therapeutic application of these compounds can be defined
either as an agent that will be used as a first line therapy in the
treatment of a disease such as cancer, as a combination therapy
where the therapeutic agents of the invention could be utilized in
conjunction with adjuvant chemotherapy (e.g, with one of the other
therapeutic agents disclosed herein), or as the therapeutic part of
a matched pair therapeutic agent. The matched pair concept refers
to a single unmetallated compound which can serve as both a
diagnostic and a therapeutic agent depending on the radiometal that
has been selected for binding to the appropriate chelate. If the
chelator cannot accommodate the desired metals appropriate
substitutions can be made to accommodate the different metal while
maintaining the pharmacology such that the behaviour of the
diagnostic compound in vivo can be used to predict the behaviour of
the radiotherapeutic compound.
7B. Optical Imaging, Sonoluminescence, Photoacoustic Imaging and
Phototherapy
In accordance with the present invention, a number of optical
parameters may be employed to determine the location of a target
with in vivo light imaging after injection of the subject with an
optically-labeled compound of the invention. Optical parameters to
be detected in the preparation of an image may include transmitted
radiation, absorption, fluorescent or phosphorescent emission,
light reflection, changes in absorbance amplitude or maxima, and
elastically scattered radiation. For example, biological tissue is
relatively translucent to light in the near infrared (NIR)
wavelength range of 650-1000 nm. NIR radiation can penetrate tissue
up to several centimeters, permitting the use of compounds of the
present invention to image target-containing tissue in vivo. The
use of visible and near-infrared (NIR) light in clinical practice
is growing rapidly. Compounds absorbing or emitting in the visible,
NIR, or long-wavelength (UV-A, >350 nm) region of the
electromagnetic spectrum are potentially useful for optical
tomographic imaging, endoscopic visualization, and
phototherapy.
A major advantage of biomedical optics lies in its therapeutic
potential. Phototherapy has been demonstrated to be a safe and
effective procedure for the treatment of various surface lesions,
both external and internal. Dyes are important to enhance signal
detection and/or photosensitizing of tissues in optical imaging and
phototherapy. Previous studies have shown that certain dyes can
localize in tumors and serve as a powerful probe for the detection
and treatment of small cancers (D). A. Bellnier et al., Murine
pharmacokinetics and antitumor efficacy of the photodynamic
sensitizer 2-[1-hexyloxyethyl]-2-devinyl pyropheophorbide-a, J.
Photochem. Photobiol., 1993, 20, pp. 55-61; G. A. Wagnieres et al.,
In vivo fluorescence spectroscopy and imaging for oncological
applications, Photochem. Photobiol., 1998, 68, pp. 603-632; J. S.
Reynolds et al., Imaging of spontaneous canine mammary tumors using
fluorescent contrast agents, Photochem. Photobiol., 1999, 70, pp.
87-94). However, these dyes do not localize preferentially in
malignant tissues.
In an exemplary embodiment, the compounds of the invention may be
conjugated with photolabels, such as optical dyes, including
organic chromophores or fluorophores, having extensive delocalized
ring systems and having absorption or emission maxima in the range
of 400-1500 nm. The compounds of the invention may alternatively be
derivatized with a bioluminescent molecule. The preferred range of
absorption maxima for photolabels is between 600 and 1000 nm to
minimize interference with the signal from hemoglobin. Preferably,
photoabsorption labels have large molar absorptivities, e.g.
>105 cm.sup.-1M.sup.-1, while fluorescent optical dyes will have
high quantum yields. Examples of optical dyes include, but are not
limited to those described in WO 98/18497, WO 98/18496, WO
98/18495, WO 98/18498, WO 98/53857, WO 96/17628, WO 97/18841, WO
96/23524, WO 98/47538, and references cited therein. For example,
the photolabels may be covalently linked directly to compounds of
the invention, such as, for example, compounds comprised of
targeting moieties and linkers of the invention or compounds
comprised of diagnostic and/or therapeutic moieties and linkers of
the invention.
Several dyes that absorb and emit light in the visible and
near-infrared region of electromagnetic spectrum are currently
being used for various biomedical applications due to their
biocompatibility, high molar absorptivity, and/or high fluorescence
quantum yields. The high sensitivity of the optical modality in
conjunction with dyes as contrast agents parallels that of nuclear
medicine, and permits visualization of organs and tissues without
the undesirable effect of ionizing radiation. Cyanine dyes with
intense absorption and emission in the near-infrared (NIR) region
are particularly useful because biological tissues are optically
transparent in this region (B. C. Wilson, Optical properties of
tissues. Encyclopedia of Human Biology, 1991, 5, 587-597). For
example, indocyanine green, which absorbs and emits in the NIR
region has been used for monitoring cardiac output, hepatic
functions, and liver blood flow (Y-L. He, H. Tanigami, H. Ueyama,
T. Mashimo, and I. Yoshiya, Measurement of blood volume using
indocyanine green measured with pulse-spectrometry: Its
reproducibility and reliability. Critical Care Medicine, 1998,
26(8), 1446-1451; J. Caesar, S. Shaldon, L. Chiandussi, et al., The
use of Indocyanine green in the measurement of hepatic blood flow
and as a test of hepatic function. Clin. Sci. 1961, 21, 43-57) and
its functionalized derivatives have been used to conjugate
biomolecules for diagnostic purposes (R. B. Mujumdar, L. A. Ernst,
S. R. Mujumdar, et al., Cyanine dye labeling reagents:
Sulfoindocyanine succinimidyl esters. Bioconjugate Chemistry, 1993,
4(2), 105-111; Linda G. Lee and Sam L. Woo. "N-Heteroaromatic ion
and iminium ion substituted: cyanine dyes for use as fluorescent
labels", U.S. Pat. No. 5,453,505; Eric Hohenschuh, et al. "Light
imaging contrast agents", WO 98/48846; Jonathan Turner, et al.
"Optical diagnostic agents for the diagnosis of neurodegenerative
diseases by means of near infra-red radiation", WO 98/22146; Kai
Licha, et al. "In-vivo diagnostic process by near infrared
radiation", WO 96/17628; Robert A. Snow, et al., Compounds, WO
98/48838.
After injection of the optically-labeled compound, the patient is
scanned with one or more light sources (e.g., a laser) in the
wavelength range appropriate for the photolabel employed in the
agent. The light used may be monochromatic or polychromatic and
continuous or pulsed. Transmitted, scattered, or reflected light is
detected via a photodetector tuned to one or multiple wavelengths
to determine the location of target-containing tissue in the
subject. Changes in the optical parameter may be monitored over
time to detect accumulation of the optically-labeled reagent at the
target site. Standard image processing and detecting devices may be
used in conjunction with the optical imaging reagents of the
present invention.
The optical imaging reagents described above may also be used for
acousto-optical or sonoluminescent imaging performed with
optically-labeled imaging agents (see, U.S. Pat. No. 5,171,298, WO
98/57666, and references therein). In acousto-optical imaging,
ultrasound radiation is applied to the subject and affects the
optical parameters of the transmitted, emitted, or reflected light.
In sonoluminescent imaging, the applied ultrasound actually
generates the light detected. Suitable imaging methods using such
techniques are described in WO 98/57666.
Various imaging techniques and reagents are described in U.S. Pat.
Nos. 6,663,847, 6,656,451, 6,641,798, 6,485,704, 6,423,547,
6,395,257, 6,280,703, 6,277,841, 6,264,920, 6,264,919, 6,228,344,
6,217,848, 6,190,641, 6,183,726, 6,180,087, 6,180,086, 6,180,085,
6,013,243, and published U.S. Patent Applications 2003185756,
20031656432, 2003158127, 2003152577, 2003143159, 2003105300,
2003105299, 2003072763, 2003036538, 2003031627, 2003017164,
2002169107, 2002164287, and 2002156117.
7C. Magnetic Resonance Imaging
The compounds of the present invention may advantageously be
conjugated with one or more paramagnetic metal chelates in order to
form a contrast agent for use in MRI. Preferred paramagnetic metal
ions have atomic numbers 21-29, 42, 44, or 57-83. This includes
ions of the transition metal or lanthanide series which have one,
and more preferably five or more, unpaired electrons and a magnetic
moment of at least 1.7 Bohr magneton. Preferred paramagnetic metals
include, but are not limited to, chromium (III), manganese (II),
manganese (III), iron (II), iron (III), cobalt (II), nickel (II),
copper (II), praseodymium (III), neodymium (III), samarium (III),
gadolinium (III), terbium (III), dysprosium (III), holmium (III),
erbium (III), europium (III) and ytterbium (III). Additionally,
compounds of the present invention may also be conjugated with one
or more superparamagnetic particles.
Gd(III) is particularly preferred for MRI due to its high
relaxivity and low toxicity, and the availability of only one
biologically accessible oxidation state. Gd(III) chelates have been
used for clinical and radiologic MR applications since 1988, and
approximately 30% of MR exams currently employ a gadolinium-based
contrast agent.
One skilled in the art will select a metal according to dose
required to detect target containing tisssue and considering other
factors such as toxicity of the metal to the subject. See, Tweedle
et al., Magnetic Resonance Imaging (2nd ed.), vol. 1, Partain et
al., eds. (W.B. Saunders Co. 1988), pp. 796-7. Generally, the
desired dose for an individual metal will be proportional to its
relaxivity, modified by the biodistribution, pharmacokinetics and
metabolism of the metal. The trivalent cation, Gd.sup.3+ is
particularly preferred for MRI contrast agents, due to its high
relaxivity and low toxicity, with the further advantage that it
exists in only one biologically accessible oxidation state, which
minimnizes undesired metabolization of the metal by a patient.
Another useful metal is Cr.sup.3+, which is relatively
inexpensive.
The paramagnetic metal chelator is a molecule having one or more
polar groups that act as a ligand for, and complex with, a
paramagnetic metal. Suitable chelators are known in the art and
include acids with methylene phosphonic-acid groups, methylene
carbohydroxamine acid groups, carboxyethylidene groups, or
carboxymethylene groups. Examples of chelators include, but are not
limited to, diethylenetriamine pentaacetic acid (DTPA),
1,4,7,10-tetraazacyclotetradecane-1,4,7,10-tetraacetic acid (DOTA),
1-substituted 1,4,7,-tricarboxymethyl 1,4,7,10 teraazacyclododecane
triacetic acid (DO3A), ethylenediaminetetraacetic acid (EDTA), and
1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA).
Additional chelating ligands are
ethylenebis-(2-hydroxy-phenylglycine) (EHPG), and derivatives
thereof, including 5-Cl-EHPG, 5Br-EHPG, 5-Me-EHPG, 5t-Bu-EHPG, and
5sec-Bu-EHPG; benzodiethylenetriamine pentaacetic acid (benzo-DTPA)
and derivatives thereof, including dibenzo-DTPA, phenyl-DTPA,
diphenyl-DTPA, benzyl-DTPA, and dibenzyl DTPA; bis-2
(hydroxybenzyl)-ethylene-diaminediacetic acid (HBED) and
derivatives thereof; the class of macrocyclic compounds which
contain at least 3 carbon atoms, more preferably at least 6, and at
least two heteroatoms (O and/or N), which macrocyclic compounds can
consist of one ring, or two or three rings joined together at the
hetero ring elements, e.g., benzo-DOTA, dibenzo-DOTA, and
benzo-NOTA, where NOTA is 1,4,7-triazacyclononane
N,N',N''-triacetic acid, benzo-TETA, benzo-DOTMA, where DOTMA is
1,4,7,10-tetraazacyclotetradecane-1,4,7,10-tetra(methyl tetraacetic
acid), and benzo-TETMA, where TETMA is
1,4,8,11-tetraazacyclotetradecane-1,4,8,11-(methyl tetraacetic
acid); derivatives of 1,3-propylenediaminetetraacetic acid (PDTA)
and triethylenetetraaminehexaacetic acid (TTHA); derivatives of
1,5,10-N,N',N''-tris(2,3-dihydroxybenzoyl)-tricatecholate (LICAM)
and 1,3,5-N,N',N''-tris(2,3-dihydroxybenzoyl) aminomethylbenzene
(MECAM). A preferred chelator for use in the present invention is
DTPA. Examples of representative chelators and chelating groups
contemplated by the present invention are described in WO 98/18496,
WO 86/06605, WO 91/03200, WO 95/28179, WO 96/23526, WO 97/36619,
PCT/US98/01473, PCT/US98/20182, and U.S. Pat. Nos. 4,899,755,
5,474,756, 5,846,519 and 6,143,274, each of which is hereby
incorporated by reference in its entirety. Use of the chelate DO3A
is particularly preferred.
As with chelators for radionuclides, chelators for paramagnetic
metals may include a spacer group as defined supra.
In general, methods disclosed herein as well as other known methods
can be used to couple the metal chelate and a compound comprising a
linker of the invention. See, e.g., WO 95/28967, WO 98/18496, WO
98/18497 and discussion therein. The present invention contemplates
linking of the chelate(s) on any position, provided the metal
chelate retains the ability to bind the metal tightly in order to
minimize toxicity. Similarly, a component of a compound of this
invention may be modified or elongated in order to generate a locus
for attachment to a metal chelate, provided such modification or
elongation does not eliminate its ability to bind the target.
MRI contrast reagents prepared according to the disclosures herein
may be used in the same manner as conventional MRI contrast
reagents. When imaging target containing tissue such as, for
example, a site of angiogenesis, certain MR techniques and pulse
sequences may be preferred to enhance the contrast of the site to
the background blood and tissues. These techniques include (but are
not limited to), for example, black blood angiography sequences
that seek to make blood dark, such as fast spin echo sequences
(see, e.g., Alexander et al., Magnetic Resonance in Medicine,
40(2): 298-310 (1998))- and flow-spoiled gradient echo sequences
(see, e.g., Edelman et al., Radiology, 177(1): 45-50 (1990)). These
methods also include flow independent techniques that enhance the
difference in contrast, such as inversion-recovery prepared or
saturation-recovery prepared sequences that will increase the
contrast between target containing tissue, such as an angiogenic
tumor, and background tissues. Finally, magnetization transfer
preparations may also improve contrast with these agents (see,
e.g., Goodrich et al, Investigative Radiology, 31(6): 323-32
(1996)).
The labeled reagent is administered to the patient in the form of
an injectable composition. The method of administering the MRI
contrast agent is preferably parenterally, meaning intravenously,
intraarterially, intrathecally, interstitially, or
intracavitarilly. For imaging active angiogenesis, intravenous or
intraarterial administration is preferred.
For MRI, it is contemplated that the subject will receive a dosage
of contrast agent sufficient to enhance the MR signal at the target
(e.g. a site of angiogenesis) at least 10%. After injection of the
compound construct including the MRI reagent, the patient is
scanned in the MRI machine to determine the location of any sites
containing the target. In therapeutic settings, upon target
localization, a cytotoxic or therapeutic agent can be immediately
administered, if necessary, and the patient can be subsequently
scanned to visualize the therapeutic effect.
In a preferred embodiment, compounds including linkers of the
invention and targeting peptides or therapeutic moieties are
conjugated to one or more paramagnetic metal chelates or one or
more superparamagnetic particles. Such compound constructs are
complexed with one or more paramagnetic metals and adminitered in a
dose sufficient to enhance the MR signal at the site at least 10%.
After injection, the patient is scanned to determine the location
of any targeted sites.
7D. Ultrasound Imaging
When ultrasound is transmitted through a substance, the acoustic
properties of the substance will depend upon the velocity of the
transmissions and the density of the substance. Changes in the
acoustic properties will be most prominent at the interface of
different substances (solids, liquids, gases). Ultrasound contrast
agents are intense sound wave reflectors because of the acoustic
differences between the agent and the surrounding tissue. Gas
containing or gas generating ultrasound contrast agents are
particularly useful because of the acoustic difference between
liquid (e.g., blood) and the gas-containing or gas generating
ultrasound contrast agent. Because of their size, ultrasound
contrast agents comprising microbubbles, microballoons, and the
like may remain for a longer time in the blood stream after
injection than other detectable moieties; thus a targeted
ultrasound agent may demonsrate superior imaging of tissue
expressing or containing the target.
In this aspect of the invention, the compound constructs of the
invention may include as the diagnostic moiety a material that is
useful for ultrasound imaging. For example, compounds comprising
linkers of the invention and a targeting moiety, another diagnostic
moiety or a therapeutic moiety, may be linked to materials employed
to form vesicles (e.g., microbubbles, microballoons, microspheres,
etc.), or emulsions containing a liquid or gas which functions as
the detectable label (e.g., an echogenic gas or material capable of
generating an echogenic gas). Materials for the preparation of such
vesicles include surfactants, lipids, sphingolipids, oligolipids,
phospholipids, proteins, polypeptides, carbohydrates, and synthetic
or natural polymeric materials. See e.g. WO 98/53857, WO 98/18498,
WO 98/18495, WO 98/18497, WO 98/18496, and WO 98/18501 incorporated
herein by reference in their entirety.
For contrast agents comprising suspensions of stabilized
microbubbles (a preferred embodiment), phospholipids, and
particularly saturated phospholipids are preferred. The preferred
gas-filled microbubbles can be prepared by means known in the art,
such as, for example, by a method described in any one of the
following patents: EP 554213, U.S. Pat. Nos. 5,413,774, 5,578,292,
EP 744962, EP 682530, U.S. Pat. Nos. 5,556,610, 5,846,518,
6,183,725, EP 474833, U.S. Pat. Nos. 5,271,928, 5,380,519,
5,531,980, 5,567,414, 5,658,551, 5,643,553, 5,911,972, 6,110,443,
6,136,293, EP 619743, U.S. Pat. Nos. 5,445,813, 5,597,549,
5,686,060, 6,187,288, and 5,908,610, each of which is incorporated
by reference herein in its entirety. In a preferred embodiment, at
least one of the phospholipid moieties has the structure described
in U.S. Pat. No. 5,686,060, which is herein incorporated by
reference in its entirety.
Examples of suitable phospholipids include esters of glycerol with
one or two molecules of fatty acids (the same or different) and
phosphoric acid, wherein the phosphoric acid residue is in turn
bonded to a hydrophilic group, such as choline, serine, inositol,
glycerol, ethanolamine, and the like groups. Fatty acids present in
the phospholipids are in general long chain aliphatic acids,
typically containing from 12 to 24 carbon atoms, preferably from 14
to 22, that may be saturated or may contain one or more
unsaturations. Examples of suitable fatty acids are lauric acid,
myristic acid, palmitic acid, stearic acid, arachidic acid, behenic
acid, oleic acid, linoleic acid, and linolenic acid. Mono esters of
phospholipid are also known in the art as the "lyso" forms of the
phospholipids.
Further examples of phospholipids are phosphatidic acids, i.e. the
diesters of glycerol-phosphoric acid with fatty acids,
sphingomyelins, i.e. those phosphatidylcholine analogs where the
residue of glycerol diester with fatty acids is replaced by a
ceramide chain, cardiolipins, i.e. the esters of
1,3-diphosphatidylglycerol with a fatty acid, gangliosides,
cerebrosides, etc.
As used herein, the term phospholipids includes either naturally
occurring, semisynthetic or synthetically prepared products that
can be employed either singularly or as mixtures. Examples of
naturally occurring phospholipids are natural lecithins
(phosphatidylcholine (PC) derivatives) such as, typically, soya
bean or egg yolk lecithins. Examples of semisynthetic phospholipids
are the partially or fully hydrogenated derivatives of the
naturally occurring lecithins. Examples of synthetic phospholipids
are e.g., dilauryloyl-phosphatidylcholine ("DLPC"),
dimyristoylphosphatidylcholine ("DMPC"),
dipalmitoyl-phosphatidylcholine ("DPPC"),
diarachidoylphosphatidylcholine ("DAPC"),
distearoyl-phosphatidylcholine ("DSPC"),
1-myristoyl-2-palmitoylphosphatidylcholine ("MPPC"),
1-palmitoyl-2-myristoylphosphatidylcholine ("PMPC"),
1-palmitoyl-2-stearoylphosphatid-ylcholine ("PSPC"),
1-stearoyl-2-palmitoylphosphatidylcholine ("SPPC"),
dioleoylphosphatidylycholine ("DOPC"), 1,2
Distearoyl-sn-glycero-3-Ethylphosphocholine (Ethyl-DSPC),
dilauryloyl-phosphatidylglycerol ("DLPG") and its alkali metal
salts, diarachidoylphosphatidylglycerol ("DAPG") and its alkali
metal salts, dimyristoylphosphatidylglycerol ("DMPG") and its
alkali metal salts, dipalmitoyl-phosphatidylglycerol ("DPPG") and
its alkali metal salts, distearolyphosphatidylglycerol ("DSPG") and
its alkali metal salts, dioleoylphosphatidylglycerol ("DOPG") and
its alkali metal salts, dimyristoyl phosphatidic acid ("DMPA") and
its alkali metal salts, dipalmitoyl phosphatidic acid ("DPPA") and
its alkali metal salts, distearoyl phosphatidic acid ("DSPA"),
diarachidoyl phosphatidic acid ("DAPA") and its alkali metal salts,
dimyristoyl phosphatidyl-ethanolamine ("DMPE"), dipalmitoyl
phosphatidylethanolamine ("DPPE"), distearoyl
phosphatidyl-ethanolamine ("DSPE"), dimyristoyl phosphatidylserine
("DMPS"), diarachidoyl phosphatidylserine ("DAPS"), dipalmitoyl
phosphatidylserine ("DPPS"), distearoylphosphatidylserine ("DSPS"),
dioleoylphosphatidylserine ("DOPS"), dipalmitoyl sphingomyelin
("DPSP"), and distearoyl sphingomyelin ("DSSP").
Other preferred phospholipids include
dipalmitoylphosphatidylcholine, dipalmitoylphosphatidic acid and
dipalmitoylphosphatidylserine. The compositions may also contain
PEG4000 and/or palmitic acid. Any of the gases disclosed herein or
known to the skilled artisan may be employed; however, inert gases,
such as SF.sub.6, or fluorocarbons, such as CF.sub.4,
C.sub.3F.sub.8 and C.sub.4F.sub.10, are preferred.
The preferred microbubble suspensions may be prepared from
phospholipids using known processes such as a freeze-drying or
spray-drying solutions of the crude phospholipids in a suitable
solvent or using the processes set forth in EP 554213, U.S. Pat.
Nos. 5,413,774, 5,578,292, EP 744962, EP 682530, U.S. Pat. Nos.
5,556,610, 5,846,518, 6,183,725, EP 474833, U.S. Pat. Nos.
5,271,928, 5,380,519, 5,531,980, 5,567,414, 5,658,551, 5,643,553,
5,911,972, 6,110,443, 6,136,293, EP 619743, U.S. Pat. Nos.
5,445,813, 5,597,549, 5,686,060, 6,187,288, and 5,908,610, each of
which is incorporated by reference herein in its entirety. Most
preferably, the phospholipids are dissolved in an organic solvent
and the solution is dried without going through a liposome
formation stage. This can be done by dissolving the phospholipids
in a suitable organic solvent together with a hydrophilic
stabilizer substance or a compound soluble both in the organic
solvent and water and freeze-drying or spray-drying the solution.
In this embodiment the criteria used for selection of the
hydrophilic stabilizer is its solubility in the organic solvent of
choice. Examples of hydrophilic stabilizer compounds soluble in
water and the organic solvent are e.g. a polymer, like polyvinyl
pyrrolidone (PVP), polyvinyl alcohol (PVA), polyethylene glycol
(PEG), etc., malic acid, glycolic acid, maltol and the like. Such
hydrophilic compounds also aid in homogenizing the microbubbles
size distribution and enhance stability under storage. Any suitable
organic solvent may be used as long as its boiling point is
sufficiently low and its melting point is sufficiently high to
facilitate subsequent drying. Typical organic solvents include, for
example, dioxane, cyclohexanol, tertiary butanol,
tetrachlorodifluoro ethylene (C.sub.2Cl.sub.4F.sub.2) or
2-methyl-2-butanol however, 2-methyl-2-butanol and
C.sub.2Cl.sub.4F.sub.2 are preferred.
Prior to formation of the suspension of microbubbles by dispersion
in an aqueous carrier, the freeze-dried or spray-dried phospholipid
powders are contacted with air or another gas. When contacted with
the aqueous carrier the powdered phospholipids whose structure has
been disrupted will form lamellarized or laminarized segments that
will stabilize the microbubbles of the gas dispersed therein. This
method permits production of suspensions of microbubbles that are
stable even when stored for prolonged periods and are obtained by
simple dissolution of the dried laminarized phospholipids (which
have been stored under a desired gas) without shaking or any
violent agitation.
Alternatively, microbubbles can be prepared by suspending a gas
into an aqueous solution at high agitation speed, as disclosed e.g.
in WO 97/29783. A further process for preparing microbubbles is
disclosed in co-pending European patent application no. 03002373,
herein incorporated by reference, which comprises preparing an
emulsion of an organic solvent in an aqueous medium in the presence
of a phospholipid andsubsequently lyophilizing said emulsion, after
optional washing and/or filtration steps.
Additives known to those of ordinary skill in the art can be
included in the suspensions of stabilized microbubbles. For
instance, non-film forming surfactants, including polyoxypropylene
glycol and polyoxyethylene glycol and similar compounds, as well as
various copolymers thereof; fatty acids such as myristic acid,
palmitic acid, stearic acid, arachidic acid or their derivatives,
ergosterol, phytosterol, sitosterol, lanosterol, tocopherol, propyl
gallate, ascorbyl palmitate and butylated hydroxytoluene may be
added. The amount of these non-film forming surfactants is usually
up to 50% by weight of the total amount of surfactants but
preferably between 0 and 30% by weight.
Other gas containing suspensions include those disclosed in, for
example, U.S. Pat. No. 5,798,091 and WO 97/29783, incorporated
herein by reference in their entirety. These agents may be prepared
as described in U.S. Pat. No. 5,798,091 or WO97/29783, each of
which is incorporated by reference in its entirety.
Another preferred ultrasound contrast agent comprises
microballoons. The term "microballoon" refers to gas filled bodies
with a material boundary or envelope. More on microballoon
formulations and methods of preparation may be found in EP-A-0 324
938 U.S. Pat. Nos. 4,844,882; 5,711,933; 5,840,275; 5,863,520;
6,123,922; 6,200,548; 4,900,540; 5,123,414; 5,230,882; 5,469,854;
5,585,112; 4,718,433; 4,774,958; WO 9501187; U.S. Pat. Nos.
5,529,766; 5,536,490 and 5,990,263, each of which is incorporated
herein by reference in its entirety.
The preferred microballoons have an envelope including a
biodegradable physiologically compatible polymer or, a
biodegradable solid lipid. The polymers useful for the preparation
of the microballoons of the present invention can be selected from
the biodegradable physiologically compatible polymers, such as any
of those described in any of the following patents: EP 458745, U.S.
Pat. Nos. 5,711,933, 5,840,275, EP 554213, U.S. Pat. Nos. 5,413,774
and 5,578,292, the entire contents of which are incorporated herein
by reference. In particular, the polymer can be selected from
biodegradable physiologically compatible polymers, such as
polysaccharides of low water solubility, polylactides and
polyglycolides and their copolymers, copolymers of lactides and
lactones such as .epsilon.-caprolactone, .gamma.-valerolactone and
polypeptides. Other suitable polymers include poly(ortho)esters
(see e.g., U.S. Pat. Nos. 4,093,709; 4,131,648; 4,138,344;
4,180,646); polylactic and polyglycolic acid and their copolymers,
for instance DEXON (see J. Heller, Biomaterials 1 (1980), 51;
poly(DL-lactide-co-.epsilon.-caprolactone),
poly(DL-lactide-co-.gamma.-valerolactone),
poly(DL-lactide-co-.gamma.-butyrolactone), polyalkylcyanoacrylates;
polyamides, polyhydroxybutyrate; polydioxanone;
poly-.beta.-aminoketones (A. S. Angeloni, P. Ferruti, M. Tramontini
and M. Casolaro, The Mannich bases in polymer synthesis: 3.
Reduction of poly(beta-aminoketone)s to poly(gamma-aminoalcohol)s
and their N-alkylation to poly(gamma-hydroxyquaternary ammonium
salt)s, Polymer 23, pp 1693-1697, 1982.); polyphosphazenes
(Allcock, Harry R. Polyphosphazenes: new polymers with inorganic
backbone atoms (Science 193(4259), 1214-19 (1976)) and
polyanhydrides. The microballoons of the present invention can also
be prepared according to the methods of WO-A-96/15815, incorporated
herein by reference, where the microballoons are made from a
biodegradable membrane comprising biodegradable lipids, preferably
selected from mono-di-, tri-glycerides, fatty acids, sterols, waxes
and mixtures thereof. Preferred lipids are di- or tri-glycerides,
e.g. di- or tri-myristin, -palmityn or -stearin, in particular
tripalmitin or tristearin.
The microballoons may employ any of the gases disclosed herein or
known to the skilled artisan; however, inert gases such as
fluorinated gases are preferred. The microballoons may be suspended
in a pharmaceutically acceptable liquid carrier with optional
additives known to those of ordinary skill in the art and
stabilizers.
Other gas-containing contrast agent formulations include
microparticles (especially aggregates of microparticles) having gas
contained therein or otherwise associated therewith (for example
being adsorbed on the surface thereof and/or contained within
voids, cavities or pores therein). Methods for the preparation of
these agents are as described in EP 0122624, EP 0123235, EP
0365467, U.S. Pat. Nos. 5,558,857, 5,607,661, 5,637,289, 5,558,856,
5,137,928, WO 9521631 and WO 9313809, each of which is incorporated
herein by reference in its entirety.
Any of these ultrasound compositions should also be, as far as
possible, isotonic with blood. Hence, before injection, small
amounts of isotonic agents may be added to any of above ultrasound
contrast agent suspensions. The isotonic agents are physiological
solutions commonly used in medicine and they comprise aqueous
saline solution (0.9% NaCl), 2.6% glycerol solution, 5% dextrose
solution, etc. Additionally, the ultrasound compositions may
include standard pharmaceutically acceptable additives, including,
for example, emulsifying agents, viscosity modifiers,
cryoprotectants, lyoprotectants, bulking agents etc.
Any biocompatible gas may be used in the ultrasound contrast agents
useful in the invention. The term "gas" as used herein includes any
substances (including mixtures) substantially in gaseous form at
the normal human body temperature. The gas may thus include, for
example, air; nitrogen; oxygen; CO.sub.2; argon; xenon or krypton,
fluorinated gases (including for example, perfluorocarbons,
SF.sub.6, SeF.sub.6) a low molecular weight hydrocarbon (e.g.
containing from 1 to 7 carbon atoms), for example, an alkane such
as methane, ethane, a propane, a butane or a pentane, a cycloalkane
such as cyclopropane, cyclobutane or cyclopentene, an alkene such
as ethylene, propene, propadiene or a butene, or an alkyne such as
acetylene or propyne and/or mixtures thereof. However, fluorinated
gases are preferred. Fluorinated gases include materials which
contain at least one fluorine atom such as SF.sub.6, freons
(organic compounds containing one or more carbon atoms and
fluorine, i.e. CF.sub.4, C.sub.2F.sub.6, C.sub.3F.sub.8,
C.sub.4F.sub.8, C.sub.4F.sub.10, CBrF.sub.3, CCI.sub.2F.sub.2,
C.sub.2CIF.sub.5, and CBrClF.sub.2) and perfluorocarbons. The term
perfluorocarbon refers to compounds containing only carbon and
fluorine atoms and includes, in particular, saturated, unsaturated,
and cyclic perfluorocarbons. The saturated perfluorocarbons, which
are usually preferred, have the formula C.sub.nF.sub.n+2, where n
is from 1 to 12, preferably from 2 to 10, most preferably from 3 to
8 and even more preferably from 3 to 6. Suitable perfluorocarbons
include, for example, CF.sub.4, C.sub.2F.sub.6, C.sub.3F.sub.8,
C.sub.4F.sub.8, C.sub.4F.sub.10, C.sub.5F.sub.12, C.sub.6F.sub.12,
C.sub.7F.sub.14, C.sub.8F.sub.18, and C.sub.9F.sub.20. Most
preferably the gas or gas mixture comprises SF.sub.6 or a
perfluorocarbon selected from the group consisting of
C.sub.3F.sub.8 C.sub.4F.sub.8, C.sub.4F.sub.10, C.sub.5F.sub.12,
C.sub.6F.sub.12, C.sub.7F.sub.14, C.sub.8F.sub.18, with
C.sub.4F.sub.10 being particularly preferred. See also WO 97/29783,
WO 98/53857, WO 98/18498, WO 98/18495, WO 98/18496, WO098/18497, WO
98/18501, WO 98/05364, and WO 98/17324.
In certain circumstances it may be desirable to include a precursor
to a gaseous substance (e.g. a material that is capable of being
converted to a gas in vivo, often referred to as a "gas
precursor"). Preferably the gas precursor and the gas it produces
are physiologically acceptable. The gas precursor may be
pH-activated, photo-activated, temperature activated, etc. For
example, certain perfluorocarbons may be used as temperature
activated gas precursors. These perfluorocarbons, such as
perfluoropentane, have a liquid/gas phase transition temperature
above room temperature (or the temperature at which the agents are
produced and/or stored) but below body temperature; thus, they
undergo a phase shift and are converted to a gas within the human
body.
As discussed, the gas can include a mixture of gases. The following
combinations are particularly preferred gas mixtures: a mixture of
gases (A) and (B) in which, at least one of the gases (B), present
in an amount of between 0.5-41% by vol., has a molecular weight
greater than 80 daltons and is a fluorinated gas and (A) is
selected from the group consisting of air, oxygen, nitrogen, carbon
dioxide and mixtures thereof, the balance of the mixture being gas
A.
Since ultrasound vesicles may be larger than the other detectable
labels described herein, they may be conjugated to a plurality of
compound constructs in order to increase the targeting efficiency
of the agent. Attachment to the ultrasound contrast agents
described above (or known to those skilled in the art) may be via a
covalent bond between a linkler of the invention and the material
used to make the vesicle or via a linker, as described
previously.
A number of methods may be used to prepare suspensions of
microbubbles conjugated to compounds. For example, one may prepare
maleimide-derivatized microbubbles by incorporating 5% (w/w) of
N-MPB-PE
(1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-4-(p-maleimido-phenyl
butyramide), (Avanti Polar-Lipids, Inc) in the phospholipid
formulation. Then, solutions of mercaptoacetylated compounds of the
invention (10 mg/mL in DMF), which have been incubated in
deacetylation solution (50 mM sodium phosphate, 25 mM EDTA, 0.5 M
hydroxylamine HCl, pH 7.5) are added to the maleimide-activated
microbubble suspension. After incubation in the dark, under gentle
agitation, the compound conjugated microbubbles may be purified by
centrifugation.
Compounds that can be used for derivatization of microbubbles
typically include the following components: (a) a hydrophobic
portion, compatible with the material forming the envelope of the
microbubble or of the microballoon, in order to allow an effective
incorporation of the compound in the envelope of the vesicle; said
portion is represented typically by a lipid moiety (dipalmitin,
distearoyl); and (b) a spacer (typically PEGs of different
molecular weights), which may be optional in some cases
(microbubbles may for instance present difficulties to be freeze
dried if the spacer is too long e.g) or preferred in some others
(e.g. peptides may be less active when conjugated to a microballoon
with short spacers); and (c) a reactive group capable of reacting
with a corresponding reacting moiety on the peptide to be
conjugated (e.g. maleimido with the --SH group of cysteine).
Alternatively, compounds conjugated to microbubbles may be prepared
using biotin/avidin. For example, avidin-conjugated microbubbles
may be prepared using a maleimide-activated phospholipid
microbubble suspension, prepared as described above, which is added
to mercaptoacetylated-avidin (which has been incubated with
deacetylation solution). Biotinylated compounds of the invention
are then added to the suspension of avidin-conjugated microbubbles,
yielding a suspension of microbubbles conjugated to the
compounds.
Unless it contains a hyperpolarized gas, known to require special
storage conditions, the lyophilized residue may be stored and
transported without need of temperature control of its environment
and in particular it may be supplied to hospitals and physicians
for on site formulation into a ready-to-use administrable
suspension without requiring such users to have special storage
facilities. Preferably in such a case it can be supplied in the
form of a two-component kit which can include two separate
containers or a dual-chamber container. In the former case
preferably the container is a conventional septum-sealed vial,
wherein the vial containing the lyophilized residue of step b) is
sealed with a septum through which the carrier liquid may be
injected using an optionally prefilled syringe. In such a case the
syringe used as the container of the second component is also used
then for injecting the contrast agent. In the latter case,
preferably the dual-chamber container is a dual-chamber syringe and
once the lyophilizate has been reconstituted and then suitably
mixed or gently shaken, the container can be used directly for
injecting the contrast agent. In both cases means for directing or
permitting application of sufficient bubble forming energy into the
contents of the container are provided. However, as noted above, in
the stabilised contrast agents according to the invention the size
of the gas microbubbles is substantially independent of the amount
of agitation energy applied to the reconstituted dried product.
Accordingly, no more than gentle hand shaking is generally required
to give reproducible products with consistent microbubble size.
It can be appreciated by one of ordinary skill in the art that
other two-chamber reconstitution systems capable of combining the
dried powder with the aqueous solution in a sterile manner are also
within the scope of the present invention. In such systems, it is
particularly advantageous if the aqueous phase can be interposed
between the water-insoluble gas and the environment, to increase
shelf life of the product. Where a material necessary for forming
the contrast agent is not already present in the container (e.g. a
targeting ligand to be linked to the phospholipid during
reconstitution), it can be packaged with the other components of
the kit, preferably in a form or container adapted to facilitate
ready combination with the other components of the kit.
No specific containers, vial or connection systems are required;
the present invention may use conventional containers, vials and
adapters. The only requirement is a good seal between the stopper
and the container. The quality of the seal, therefore, becomes a
matter of primary concern; any degradation of seal integrity could
allow undesirable substances to enter the vial. In addition to
assuring sterility, vacuum retention is essential for products
stoppered at ambient or reduced pressures to assure safe and proper
reconstitution. As to the stopper, it may be a compound or
multicomponent formulation based on an elastomer, such as
poly(isobutylene) or butyl rubber.
Ultrasound imaging techniques which may be used in accordance with
the present invention include known techniques, such as color
Doppler, power Doppler, Doppler amplitude, stimulated acoustic
imaging, and two- or three-dimensional imaging techniques. Imaging
may be done in harmonic (resonant frequency) or fundamental modes,
with the second harmonic preferred.
In ultrasound applications the contrast agents formed by
phospholipid stabilized microbubbles may, for example, be
administered in doses such that the amount of phospholipid injected
is in the range 0.1 to 200 .mu.g/kg body weight, preferably from
about 0.1 to 30 .mu.g/kg. Microballoon-containing contrast agents
are typically administered in doses such that the amount of
wall-forming polymer or lipid is from about 10 .mu.g/kg to about 20
mg/kg of body weight.
In a preferred embodiment, the ultrasound contrast agents described
herein are conjugated to one or more compounds comprised of
compounds of the invention (e.g., linkers of the invention) and
targeting peptides, or other diagnostic moieties or therapeutic
moieties. The targeted ultrasound contrast agents will localize at
sites of tissue expressing the target and may be used to image
and/or treat such tissue.
7E. Radiotherapy
Radioisotope therapy involves the administration of a radiolabeled
compound in sufficient quantity to damage or destroy the targeted
tissue. After administration of the compound (by e.g. intravenous,
subcutaneous, or intraperitonal injection), the radiolabeled
pharmaceutical localizes preferentially at the disease site (in
this instance, tumor or other tissue that expresses the target).
Once localized, the radiolabeled compound then damages or destroys
the diseased tissue with the energy that is released during the
radioactive decay of the isotope that is administered.
The design of a successful radiotherapeutic involves several
critical factors: 1. selection of an appropriate targeting group to
deliver the radioactivity to the disease site; 2. selection of an
appropriate radionuclide that releases sufficient energy to damage
that disease site, without substantially damaging adjacent normal
tissues; and 3. selection of an appropriate combination of the
targeting group and the radionuclide without adversely affecting
the ability of this conjugate to localize at the disease site. For
radiometals, this often involves a chelating group that coordinates
tightly to the radionuclide, combined with a linker that couples
said chelate to the targeting group, and that affects the overall
biodistribution of the compound to maximize uptake in target
tissues and minimizes uptake in normal, non-target organs.
The present invention provides radiotherapeutic agents that satisfy
all three of the above criteria, through proper selection of
targeting group, radionuclide, metal chelate and linker.
Radiotherapeutic agents may contain a chelated 3+metal ion from the
class of elements known as the lanthanides (elements of atomic
number 57-71) and their analogs (i.e. M.sup.3+ metals such as
yttrium and indium). Typical radioactive metals in this class
include the isotopes 90-Yttrium, 111-Indium, 149-Promethium,
153-Samarium, 166-Dysprosium, 166-Holmium, 175-Ytterbium, and
177-Lutetium. All of these metals (and others in the lanthanide
series) have very similar chemistries, in that they remain in the
+3 oxidation state, and prefer to chelate to ligands that bear hard
(oxygen/nitrogen) donor atoms, as typified by derivatives of the
well known chelate DTPA (diethylenetriaminepentaacetic acid) and
polyaza-polycarboxylate macrocycles such as DOTA
(1,4,7,10-tetrazacyclododecane-N,N',N'',N'''-tetraacetic acid and
its close analogs. The structures of these chelating ligands, in
their fully deprotonated form are shown below.
TABLE-US-00002 DTPA ##STR00005## DOTA ##STR00006##
These chelating ligands encapsulate the radiometal by binding to it
via multiple nitrogen and oxygen atoms, thus preventing the release
of free (unbound) radiometal into the body. This is important, as
in vivo dissociation of 3.sup.+ radiometals from their chelate can
result in uptake of the radiometal in the liver, bone and spleen
[Brechbiel M W, Gansow O A, "Backbone-substituted DTPA ligands for
.sup.90Y radioimmunotherapy", Bioconj. Chem. 1991; 2: 187-194; Li,
WP, Ma D S, Higginbotham C, Hoffman T, Ketring A R, Cutler C S,
Jurisson, S S, "Development of an in vitro model for assessing the
in vivo stability of lanthanide chelates." Nucl. Med. Biol. 2001;
28(2): 145-154; Kasokat T, Urich K. Arzneim.-Forsch,
"Quantification of dechelation of gadopentetate dimeglumine in
rats". 1992; 42(6): 869-76]. Unless one is specifically targeting
these organs, such non-specific uptake is highly undesirable, as it
leads to non-specific irradiation of non-target tissues, which can
lead to such problems as hematopoietic suppression due to
irradiation of bone marrow.
For radiotherapy applications, any of the chelators for therapeutic
radionuclides disclosed herein may be used. However, forms of the
DOTA chelate [Tweedle M F, Gaughan G T, Hagan J T,
"1-Substituted-1,4,7-triscarboxymethyl-1,4,7,10-tetraazacyclododecane
and analogs." U.S. Pat. No. 4,885,363, Dec. 5, 1989] are
particularly preferred, as the DOTA chelate is expected to
de-chelate less in the body than DTPA or other linear chelates.
General methods for coupling DOTA-type macrocycles to targeting
groups through a linker (e.g. by activation of one of the
carboxylates of the DOTA to form an active ester, which is then
reacted with an amino group on the linker to form a stable amide
bond), are known to those skilled in the art. (See e.g. Tweedle et
al. U.S. Pat. No. 4,885,363). Coupling can also be performed on
DOTA-type macrocycles that are modified on the backbone of the
polyaza ring.
The selection of a proper nuclide for use in a particular
radiotherapeutic application depends on many factors, including: a.
Physical half-life--This should be long enough to allow synthesis
and purification of the radiotherapeutic construct from radiometal
and conjugate, and delivery of said construct to the site of
injection, without significant radioactive decay prior to
injection. Preferably, the radionuclide should have a physical
half-life between about 0.5 and 8 days. b. Energy of the
emission(s) from the radionuclide--Radionuclides that are particle
emitters (such as alpha emitters, beta emitters and Auger electron
emitters) are particularly useful, as they emit highly energetic
particles that deposit their energy over short distances, thereby
producing highly localized damage. Beta emitting radionuclides are
particularly preferred, as the energy from beta particle emissions
from these isotopes is deposited within 5 to about 150 cell
diameters. Radiotherapeutic agents prepared from these nuclides are
capable of killing diseased cells that are relatively close to
their site of localization, but cannot travel long distances to
damage adjacent normal tissue such as bone marrow. c. Specific
activity (i.e. radioactivity per mass of the
radionuclide)--Radionuclides that have high specific activity (e.g.
generator produced 90-Y, 111-In, 177-Lu) are particularly
preferred. The specific activity of a radionuclide is determined by
its method of production, the particular target that is used to
produce it, and the properties of the isotope in question.
Many of the lanthanides and lanthanoids include radioisotopes that
have nuclear properties that make them suitable for use as
radiotherapeutic agents, as they emit beta particles. Some of these
are listed in the table below.
TABLE-US-00003 Approximate range of b- particle Half-Life Max
b-energy Gamma energy (cell Isotope (days) (MeV) (keV) diameters)
.sup.149-Pm 2.21 1.1 286 60 .sup.153-Sm 1.93 0.69 103 30
.sup.166-Dy 3.40 0.40 82.5 15 .sup.166-Ho 1.12 1.8 80.6 117
.sup.175-Yb 4.19 0.47 396 17 .sup.177-Lu 6.71 0.50 208 20 .sup.90-Y
2.67 2.28 -- 150 .sup.111-In 2.810 Auger electron 173, 247 <5
.mu.m emitter Pm: Promethium, Sm: Samarium, Dy: Dysprosium, Ho:
Holmium, Yb: Ytterbium, Lu: Lutetium, Y: Yttrium, In: Indium
Methods for the preparation of radiometals such as beta-emitting
lanthanide radioisotopes are known to those skilled in the art, and
have been described elsewhere [e.g., Cutler C S, Smith C J,
Ehrhardt G J.; Tyler T T, Jurisson S S, Deutsch E. "Current and
potential therapeutic uses of lanthanide radioisotopes." Cancer
Biother. Radiopharm. 2000; 15(6): 531-545]. Many of these isotopes
can be produced in high yield for relatively low cost, and many
(e.g. .sup.90-Y, .sup.149-Pm, .sup.177-Lu) can be produced at close
to carrier-free specific activities (i.e. the vast majority of
atoms are radioactive). Since non-radioactive atoms can compete
with their radioactive analogs for binding to receptors on the
target tissue, the use of high specific activity radioisotope is
important, to allow delivery of as high a dose of radioactivity to
the target tissue as possible.
Radiotherapeutic derivatives of the invention containing
beta-emitting isotopes of rhenium (.sup.186-Re and .sup.188-Re) are
also particularly preferred.
8. Dosages And Additives
Proper dose schedules for the compounds of the present invention
are known to those skilled in the art. The compounds can be
administered using many methods which include, but are not limited
to, a single or multiple IV or IP injections. For
radiopharmaceuticals of the invention, one administers a quantity
of radioactivity that is sufficient to permit imaging, or in the
case of radiotherapy, to cause damage or ablation of the targeted
GRP-R bearing tissue, but not so much that substantive damage is
caused to non-target (normal tissue). The quantity and dose
required for scintigraphic imaging is discussed supra. The quantity
and dose required for radiotherapy is also different for different
constructs, depending on the energy and half-life of the isotope
used, the degree of uptake and clearance of the agent from the body
and the mass of the tumor. In general, doses can range from a
single dose of about 30-50 mCi to a cumulative dose of up to about
3 Curies.
The optical imaging compounds of the present invention can be
administered to a patient alone or as part of a composition that
contains other components such as excipients, diluents, radical
scavengers, stabilizers, and carriers, all of which are well-known
in the art. The optical imaging compounds can be administered to
patients either intravenously or intraperitoneally. The amount of
compound administered will typically be in the range of
approximately 0.01 mg/kg to 10.0 mg/kg of body weight of the
patient.
In ultrasound applications the contrast agents formed by
phospholipid stabilized microbubbles may, for example, be
administered in doses such that the amount of phospholipid injected
is in the range 0.1 to 200 .mu.g/kg body weight, preferably from
about 0.1 to 30 .mu.g/kg. Microballoon-containing contrast agents
are typically administered in doses such that the amount of
wall-forming polymer or lipid is from about 10 .mu.g/kg to about 20
mg/kg of body weight.
For MRI, it is contemplated that the subject will receive a dosage
of contrast agent sufficient to enhance the MR signal at the target
at least 10%. After injection of the compound including the MRI
reagent, the patient is scanned in the MRI machine to determine the
location of any sites containing the target. In therapeutic
settings, upon target localization, a cytotoxic or therapeutic
agent can be immediately administered, if necessary, and the
patient can be subsequently scanned to visualize the therapeutic
effect.
The pharmaceutical compositions of the invention can include
physiologically acceptable buffers, and typical additives,
excipients, etc. In the case of radiopharmaceuticals, compositions
of the invention can require radiation stabilizers to prevent
radiolytic damage to the compound prior to injection. Radiation
stabilizers are known to those skilled in the art, and may include,
for example, para-aminobenzoic acid, ascorbic acid, gentistic acid
and the like. Particularly preferred stabilizers and formulations
are discussed in copending provisional application U.S. Ser. No.
60/489,850.
A single or multi-vial kit that contains all of the components
needed to prepare the diagnostic or therapeutic agents of this
invention is an integral part of this invention. In the case of
radiopharmaceuticals of the invention, such kits will generally
include all of the components needed to prepare the
radiopharmaceutical except the radionuclide.
For example, a single-vial kit for preparing a radiopharmaceutical
of the invention preferably contains a chelator/linker/targeting
peptide conjugate of the formula M-N-O-P-Q, a source of stannous
salt (if reduction is required, e.g., when using technetium), or
other pharmaceutically acceptable reducing agent, and is
appropriately buffered with pharmaceutically acceptable acid or
base to adjust the pH to a value of about 3 to about 9. The
quantity and type of reducing agent used will depend highly on the
nature of the exchange complex to be formed. The proper conditions
are well known to those that are skilled in the art. It is
preferred that the kit contents be in lyophilized form. Such a
single vial kit may optionally contain labile or exchange ligands
such as glucoheptonate, gluconate, mannitol, malate, citric or
tartaric acid and can also contain reaction modifiers: such as
diethylenetriamine-pentaacetic acid (DPTA), ethylenediamine
tetraacetic acid (EDTA), or .alpha., .beta., or
.gamma.-cyclodextrin that serve to improve the radiochemical purity
and stability of the final product. The kit may also contain
stabilizers, bulking agents such as mannitol, that are designed to
aid in the freeze-drying process, and other additives known to
those skilled in the art.
A multi-vial kit preferably contains the same general components
but employs more than one vial in reconstituting the
radiopharmaceutical. For example, one vial may contain all of the
ingredients that are required to form a labile Tc(V) complex on
addition of pertechnetate (e.g. the stannous source or other
reducing agent). Pertechnetate is added to this vial, and after
waiting an appropriate period of time, the contents of this vial
are added to a second vial that contains the chelator and targeting
peptide, as well as buffers' appropriate to adjust the pH to its
optimal value. After a reaction time of about 5 to 60 minutes, the
complexes of the present invention are formed. It is advantageous
that the contents of both vials of this multi-vial kit be
lyophilized. As above, reaction modifiers, exchange ligands,
stabilizers, bulking agents, etc. may be present in either or both
vials.
General Preparation of Compounds
The compounds of the present invention can be prepared by various
methods depending upon the selected diagnostic or therapeutic
moiety. The peptide portion of the compound can be most
conveniently prepared by techniques generally established and known
in the art of peptide synthesis, such as the solid-phase peptide
synthesis (SPPS) approach. Because it is amenable to solid phase
synthesis, employing alternating FMOC protection and deprotection
is the preferred method of making short peptides. Recombinant DNA
technology is preferred for producing proteins and long fragments
thereof.
Solid-phase peptide synthesis (SPPS) involves the stepwise addition
of amino acid residues to a growing peptide chain that is linked to
an insoluble support or matrix, such as polystyrene. The C-terminal
residue of the peptide is first anchored to a commercially
available support with its amino group protected with an
N-protecting agent such as a t-butyloxycarbonyl group (Boc) or a
fluorenylmethoxycarbonyl (Fmoc) group. The amino protecting group
is removed with suitable deprotecting agents such as TPA in the
case of Boc or piperidine for Fmoc and the next amino acid residue
(in N-protected form) is added with a coupling agent such as
dicyclocarbodiimide (DCC). Upon formation of a peptide bond, the
reagents are washed from the support. After addition of the final
residue, the peptide is cleaved from the support with a suitable
reagent such as trifluoroacetic acid (TFA) or hydrogen fluoride
(HF).
EXAMPLES
The following examples are provided as examples of different
methods which can be used to prepare various compounds of the
present invention. Within each example, there are compounds
identified in single bold capital letter (e.g., A, B, C), which
correlate to the same labeled corresponding compounds in the
drawings identified. Examples I to VII are prophetic.
General Experimental Description
A. Definitions of Abbreviations Used
The following common abbreviations are used throughout this
specification: 1,1-dimethylethoxycarbonyl (Boc or Boc);
9-fluorenylmethyloxycarbonyl (Fmoc); 1-hydroxybenozotriazole
(HOBT); N,N'-diisopropylcarbodiimide (DIC); N-methylpyrrolidinone
(NNT); acetic anhydride (Ac.sub.2O);
(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl (iv-Dde);
trifluoroacetic acid (TFA); Reagent B
(TFA:H.sub.2O:phenol:triisopropylsilane, 88:5:5:2);
diisopropylethylamine (DIEA);
O-(1H-benzotriazole-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate (HBTU);
O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium
hexafluorphosphate (TU); N-hydroxysuccinimide (NHS); solid phase
peptide synthesis (SPPS); dimethylsulfoxide (DMSO); dichloromethane
(DCM); dimethylformamide (DMF); dimethylacetamide (DMA);
isobutylchloroformate (IBCF)
1,4,7,10-tetraazacyclotetradecane-1,4,7,10-tetraacetic acid (DOTA);
Triisopropylsilane (TIPS);
1,4,7,10-tetraazacyclotetradecane-1,4,7,10-tetraacetic acid (DOTA)
(1R)-1-[1,4,7,10-tetraaza-4,7, 10-tris(carboxymethyl)cyclododecyl]
ethane-1,2-dicarboxylic acid (CMDOTA); fetal bovine serum (FBS);
human serum albumin (HSA); human prostate cancer cell line (PC3);
radiochemical purity (RCP); high performance liquid chromatography
(HPLC), and magnetic resonance imaging (MRI).
B. Materials
The Fmoc-protected amino acids used were purchased from
Nova-Biochem (San Diego, Calif., USA), Advanced Chem Tech
(Louisville, Ky., USA), Chem-Impex International (Wood Dale Ill.,
USA), and Multiple Peptide Systems (San Diego, Calif., USA). Other
chemicals, reagents and adsorbents required for the syntheses were
procured from Aldrich Chemical Co. (Milwaukee, Wis., USA) and VWR
Scientific Products (Bridgeport, N.J., USA). Solvents for peptide
synthesis were obtained from Pharmco Co. (Brookfield Conn., USA).
Columns for HPLC analysis and purification were obtained from
Waters Co. (Milford, Mass., USA). Experimental details are given
below for those that were not commercially available.
C. Instrumentation for Peptide Synthesis
Peptides were prepared using an Advanced ChemTech 496.OMEGA. MOS
synthesizer, an Advanced ChemTech 357 FBS synthesizer and/or by
manual peptide synthesis. However the protocols for iterative
deprotection and chain extension employed were the same for
all.
D. Instrumentation Employed for Analysis and Purification
Analytical HPLC was performed using a Shimadzu-LC-10A dual pump
gradient analytical LC system employing Shimadzu-ClassVP software
version 4.1 for system control, data acquisition, and post run
processing. Mass spectra were acquired on a Hewlett-Packard Series
1100 MSD mass spectrometer interfaced with a Hewlett-Packard Series
1100 dual pump gradient HPLC system fitted with an Agilent
Technologies 1100 series autosampler fitted for either direct flow
injection or injection onto a Waters Associates XMerra MS C18
column (4.6 mm.times.50 mm, 5 .mu. particle, 120.ANG. pore). The
instrument was driven by a HP Kayak workstation using `MSD Anyone`
software for sample submission and HP Chemstation software for
instrument control and data acquisition. In most-cases the samples
were introduced via direct injection using a 5 .mu.L injection of
sample solution at a concentration of 1 mg/mL and analyzed using
positive ion electrospray to obtain m/e and m/z (multiply charged)
ions for confirmation of structure. .sup.1H-NMR spectra were
obtained on a Varian Innova spectrometer at 500 MHz. .sup.13C-NMR
spectra were obtained on the same instrument at 125.73 MHz.
Generally the residual .sup.1H absorption, or in the case of
.sup.13C-NMR, the .sup.13C absorption of the solvent employed, was
used as an internal reference; in other cases tetramethylsilane
(.delta.=0.00 ppm) was employed. Resonance values are given in
.delta. units. Micro-analysis data was obtained from Quantitative
Technologies Inc. Whitehouse N.J. Preparative HPLC was performed on
a Shimadzu-LC-8A dual pump gradient preparative HPLC system
employing Shimadzu-ClassVP software version 4.3 for system control,
data acquisition, fraction collection and post run processing
E. Solid Support for Peptide Synthesis:
Rink Amide-Novagel HL resin (0.6 mmol/g) was used as the solid
support.
F. Coupling Procedure:
In a typical experiment, the first amino acid was loaded onto 0.1 g
of the resin (0.06 mmol). The appropriate Fmoc-amino acid in NMo
(0.25M solution; 0.960 mL was added to the resin followed by
N-hydroxybenzotriazole (0.5M in NMP; 0.48 mL)) and the reaction
block (in the case of automated peptide synthesis) or individual
reaction vessel (in the case of manual peptide synthesis) was
shaken for about 2 min. To the above mixture,
diisopropylcarbodiimide (0.5M in NMP; 0.48 mL) was added and the
reaction mixture was shaken for 4 h at ambient temperature. Then
the reaction block or the individual reaction vessel was purged of
reactants by application of a positive pressure of dry
nitrogen.
G. Washing Procedure:
Each well of the reaction block was filled with 1.2 mL of NMP and
the block was shaken for 5 min. The solution was drained under
positive pressure of nitrogen. This procedure was repeated three
times. The same procedure was used, with an appropriate volume of
NMP, in the case of manual synthesis using individual vessels.
H. Removal of Fmoc Group:
The resin containing the Fmoc-protected amino acid was treated with
1.5 mL of 20% piperidine in DMF (v/v) and the reaction block or
individual manual synthesis vessel was shaken for 15 min. The
solution was drained from the resin. This procedure was repeated
once and the resin was washed employing the washing procedure
described above.
I. Final Coupling of Ligand (DOTA and CMDOTA):
The N-terminal amino group of the resin bound peptide linker
construct was deblocked and the resin was washed. A 0.25M solution
of the desired ligand and HBTU in NMP was made, and was treated
with a two-fold equivalency of DIEA. The resulting solution of
activated ligand was added to the resin (1.972 mL; 0.48 mmol) and
the reaction mixture was shaken at ambient temperature for 24-30 h.
The solution was drained and the resin was washed. The final wash
of the resin was conducted with 1.5 mL dichloromethane
(3.times.).
J. Deprotection and Purification of the Final Peptide:
A solution of reagent B (2 mL; 88:5:5:2-TFA:Phenol:Water:TIPS) was
added to the resin and the reaction block or individual vessel was
shaken for 4.5 h at ambient temperature. The resulting solution
containing the deprotected peptide was drained into a vial. This
procedure was repeated two more times with 1 mL of reagent B. The
combined filtrate was concentrated under reduced pressure using a
Genevac HT-12 series II centrifugal concentrator. The residue in
each vial was then triturated with 2 mL of Et.sub.2O and the
supernatant was decanted. This procedure was repeated twice to
provide the peptides as colorless solids. The crude peptides were
dissolved in water/acetonitrile and purified using either a Waters
XTerra MS C18 column (50 mm.times.19 mm, 5 micron particle size,
120.ANG. pore size) or a Waters-YMC C18 ODS column (250 mm.times.30
mm i.d., 10 micron particle size. 120 .ANG. pore size). The
fractions with the products were collected and analyzed by HPLC.
The fractions with >95% purity were pooled and the peptides
isolated by lyophilization. Conditions for Preparative HPLC (Waters
XTerra Column): Elution rate: 50 mL/min Detection: UV, X=220 nm
Eluent A: 0.1% aq. TFA; Solvent B: Acetonitrile (0.1% TFA).
Conditions for HPLC Analysis: Column: Waters XTerra (Waters Co.;
4.6.times.50 mm; MS C18; 5 micron particle, 120 .ANG. pore).
Elution rate: 3 mL/min; Detection: UV, .lamda.=220 nm. Eluent
A:0.1% aq. TFA; Solvent B: Acetonitrile (0.1% TFA).
Example I
FIG. 1
Synthesis of
4-[[(3.beta.,5.beta.,12.alpha.)-23-[1,1-dimethylethane-1-(oxycarbonyl)]-1-
2-hydroxy-24-norcholan-3-yl]amino]-4-oxobutanoic acid
N-hydroxysuccinimidyl ester (Compound A-OSu)
Deoxycholic acid is converted into the corresponding t-butyl ester
1 according to the procedure for the esterification of cholic acid
reported by R. P. Bonar-Law et al. (J. Chem. Soc. Perkin Trans. 1,
2245, 1990). Compound 1 is transformed into the 3-amino derivative
2 applying the one pot Mitsunobu-Staudinger procedure developed by
P. L. Anelli et al. (Synth. Commun. 1998, 28, 109-117) to prepare
methyl 3 .beta.-aminodeoxycholate.
Amino ester 2 is reacted with an equimolar amount of succinic
anhydride in THF in the presence of triethylamine. After acidic (aq
HCl) work up the mixture is extracted with EtOAc. The organic phase
is evaporated to dryness and the residue purified by flash
chromatography to give derivative 3. Yield 55%.
Acid 3 is reacted with N-hydroxysuccinimide and
dicyclohexylcarbodiimide in a mixture of THF and acetonitrile at
room temperature. After precipitation of dicyclohexylurea the
mixture is filtered and evaporated to afford crude compound A-OSu
which is used in the following step without any further
purification.
Example II
FIG. 2
Synthesis of Bovine
N.sup.eB29-[4-[[(3.beta.,5.beta.,12.alpha.)-23-carboxy-12-hydroxy-24-norc-
holan-3-yl]amino]-4-oxobutanoyl]-insulin (Compound B)
N.sup..alpha.A1,N.sup..alpha.A2-dicitraconyl-insulin (bovine) is
prepared according to Naithani V. K. & Gattner H.-G.:
Hoppe-Seyler's Z. Physiol. Chem. 349, 373-384, 1968. To a solution
of N.sup..alpha.A1,N.sup..alpha.A2-dicitraconyl-insulin (14
.mu.mol) in 24 mL of dimethylformamide is added triethylamine (92.7
.mu.mol) in 1.2 mL dimethylformamide. Compound A-OSu (79 mmol) in 1
mL dimethylformamide is added and the reaction mixture allowed to
proceed for 30 min at room temperature. The reaction mixture is
acidified with 1 M acetic acid and chromatographed on Sephadex G-25
(2.times.70 cm) in 1 M acetic acid. The protein peak fractions are
pooled and lyophilized. Decitraconylation is achieved in formic
acid pH 3.5 for 72 h, followed by renewed lyophilization. The
material is fractionated by preparative isoelectric focusing in
flat-bed gel-stabilized layers according to Radola: Biochim.
Biophys. Acta 295, 412-428, 1973, except that Ultradex.TM.
(Amersham Biosciences Inc.), as washed gel medium, is used. The gel
is suspended in 5 M urea (purified over mixed bed resin) and
carrier ampholytes (2%), pH 4 to 6, were added. The slurry (210 mL)
is placed in a flat plate (Desaga/Brhikmann) (20.times.20 cm), the
sample applied 2 cm from the cathode, and focusing is achieved at
25 to 30 V/cm for 18 to 24 h on a 1-4.degree. C. cooling plate
(Hormuth/Brinkmann). Visibile (refractive) bands are formed, and
the pH in the bands is determined after dilution of a gel sample in
water. The major band corresponds to a pH of 5.1, the expected pH
for insulin with a selective modification of Lys.sup..epsilon.B29
with compound A. The material from the major band is recovered from
the gel by elution with three bed volumes of 1 M acetic acid. Urea
is removed by chromatography on Sephadex G-25 (2.5.times.70 cm) in
1 M-acetic acid and protein fractions are lyophilized. The protein
is dissolved in and loaded onto a chromatography column of
DEAE-cellulose DE52 (0.9.times.25 cm) in 7 M urea, 0.01 M Tris/HCl,
pH 8.3, at room temperature. The column is eluted with the same
buffer forming a linear gradient of NaCl (0 to 0.15 M). Fractions
of 6.1 mL are collected. The major peak is collected and desalted
on a column of Sephadex G-25 (2.5.times.70 cm) in 1 M acetic acid
and protein fractions are lyophilized. The t-butyl ester is cleaved
by dissolution in trifluoroacetic acid. After evaporation of the
trifluoroacetic acid, the protein (Compound B) is dissolved in 1 M
acetic acid and lyophilized. Analytical polyacrylamide disc gel
electrophoresis according to Davis B. J.: Ann. N.Y. Acad. Sci. 121,
404-427, 1964, demonstrates the homogeneity of the material.
Ion-spray mass spectrometry shows the correct molecular weight for
the expected Compound B. Compound B comprises a therapeutic moiety
(an insulin molecule) attached to a cholic acid derivative linker
of the invention.
Example III
FIGS. 3 and 4
A. Syntheis of
(3.beta.,5.beta.,12.alpha.)-3-[[3,5-Bis[[4-[(2,5-dioxo-1-pyrrolidinyl)oxy-
]-1,4-dioxobutyl]amino]benzoyl]amino]-12-hydroxycholan-24-oic acid
1,1-dimethylethyl ester (Compound C--(OSu).sub.2)
A solution of 3,5-dinitrobenzoyl chloride 1 (commercial product) in
ethanol free chloroform is added dropwise to a solution of
(3.beta.,5.beta.,12.alpha.)-3-amino-12-hydroxycholanoic acid
t-butyl ester 2 in ethanol free chloroform and triethylamine. After
completion of the reaction the mixture is washed with water, dried
(Na.sub.2SO.sub.4), filtered and evaporated. The crude filtrate is
purified by flash chromatography on silica gel to give pure
compound 3 which is hydrogenated with palladium (10% on activated
carbon) in ethanol to give compound 4. Compound 4 is dissolved in
dichloromethane and reacted with two molar equivalents of succinic
anhydride. The intermediate diacid is then reacted in ethanol free
chloroform with N-hydroxysuccinimide (HOSu) in the presence of
1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (EDC)
and 4-dimethylaminopyridine (DMAP). After completion of the
reaction the mixture is washed with water, dried with
Na.sub.2SO.sub.4, filtered and evaporated to give compound
5=C--(OSu).sub.2.
B. Synthesis of Bovine
1-[[(3.beta.,5.beta.,12.alpha.)-23-[(1,1-dimethyl)ethoxycarbonyl]-12-hydr-
oxy-24-norcholan-3-yl]amino]carbonyl-3,5-bis[[4-(insulin-N.sup..epsilon.B2-
9-yl)-1,4-dioxobutyl]amino]benzene (Compound D) (FIG. 4)
N.sup..alpha.A1,N.sup..alpha.A2-dicitraconyl-insulin (bovine) is
prepared according to Naithani V. K. & Gattner H.-G.:
Hoppe-Seyler's Z Physiol. Chem. 349, 373-384, 1968. To a solution
of N.sup..alpha.A1,N.sup..alpha.A2-dicitraconyl-insulin (28
.mu.mol) in 48 mL of dimethylformamide is added triethylamine (185
.mu.mol) in 2.4 mL dimethylformamide. Compound C--(OSu).sub.2 (14
.mu.mol) in 2 mL dimethylformamide is added and the reaction
mixture is allowed to proceed for 30 min at room temperature. The
reaction mixture is acidified with 1 M acetic acid and
chromatographed on Sephadex G-25 (2.times.70 cm) in 1 M acetic
acid. The protein peak fractions are pooled and lyophilized.
Decitraconylation is achieved in formic acid pH 3.5 for 72 h,
followed by renewed lyophilization. The t-butyl ester is cleaved by
dissolution in trifluoroacetic acid. After evaporation of the
trifluoroacetic acid, the protein is dissolved in 1 M acetic acid
and fractionated by chromatography on Sephadex G-50 in 1 M acetic
acid. The protein peak of the molecule with the largest size is
collected and lyophilized. The material is once more fractionated
on the same column. The fractionated protein is lyophilized.
Ion-spray mass spectrometry shows the correct molecular weight for
the expected Compound D. Compound D comprises a therapeutic moiety
(an insulin molecule) attached to a linker of the invention, which
is attached to another therapeutic moiety (an insulin
molecule).
Example IV
Labeling of Compounds B and D with .sup.125I
Labeling of Compounds B and D with .sup.125I is achieved by the
method described in Lipkin E. W., Teller D. C. & de Haen C.: J.
Biol. Chem. 261, 1694-1701, 1986 or as described herein.
Example V
Binding of Compounds B and D to Human Serum Albumin
Binding to human serum albumin is determined by ultrafiltration
according to Whitlam J. B. & Brown K. F.: J. Pharm. Sci. 70,
146-150, 1981, applying 1 nM bovine insulin solution in 0.6 mM
human serum albumin, spiked with .sup.125I-labeled insulin. 95% of
the insulin is bound. It is evident that such binding to albumin
will reduce the renal elimination of the modified insulin
(Compounds B and D) relative to that of native insulin.
Example VI
Biological Activity of Compounds B and D
It is known that N.sup..epsilon.B29-biotinylinsulin bound to a 1:1
conjugate of avidin and ferritin with three of the four biotin
binding sites occupied by biotin has the same dose-response curve
as insulin for the activation of glucose oxidation in rat
epididymal fat cells (May J. M., Williams R. H. & de Haen C.:
J. Biol. Chem. 253, 686-690, 1978). Using the fat cell assay
described therein, which involves incubation in buffers containing
serum albumin, compounds B and D are also shown to have identical
activity to native insulin. Combined with the reduced renal
elimination, this property renders Compounds B and D forms of
insulin with prolonged plasma half-life, providing improved
pharmacokinetic properties compared to compounds which do not
contain linkers of the invention.
Example VII
FIGS. 5A-B
Synthesis of .sup.111In-Labeled Insulin Deoxycholic Acid Conjugate
(Compound .sup.111In--F)
A. Synthesis of Bovine
1-[[(3.beta.,5.beta.,12.alpha.)-23-[(1,1-dimethyl)ethoxycarbonyl]-12-hydr-
oxy-24-norcholan-3-yl]amino]carbonyl-3-[[4-(insulin-N.sup..epsilon.B29-yl)-
-1,4-dioxobutyl]amino]-5-[[[[2-[[[4,7,10-tris(carboxymethyl)-1,4,7,10-tetr-
aazacyclododecyl]acetyl]amino]ethyl]amino]-1,4-dioxobutyl]amino]benzene.
(Compound F)
N.sup.1,N.sup.4,
N.sup.7-triacetato-1,4,7,10-tetraazacyclododecan-N.sup.10-2-acetamidoethy-
lamine) (Compound E) (FIG. 5A) is synthesized according to
Margerum, L.; Campion, B.; Fellmann, J. D.; Garrithy, M.;
Varadarajan, J. PCT Int. Appl. WO9528967).
N.sup..alpha.A1,N.sup..alpha.A2-dicitraconyl-insulin (bovine) is
prepared according to Naithani V. K. & Gattner H.-G.:
Hoppe-Seyler's Z Physiol. Chem. 349, 373-384, 1968. To a solution
of N.sup..alpha.A1,N.sup..alpha.A2-dicitraconyl-insulin (28
.mu.mol) in 48 mL of dimethylformamide is added triethylamine (185
.mu.mol) in 2.4 mL dimethylformamide. Compound C--(OSu).sub.2 (see
Example E) (140 .mu.mol) in 2 mL of dimethylformamide is added and
the reaction is allowed to proceed for 30 min. at room temperature.
Then compound E (280 .mu.mol) in 2 mL of dimethylformamide is added
and the reaction is allowed to proceed for 30 min. at room
temperature. The reaction mixture is acidified with 1 M acetic acid
exhaustively dialyzed against 1 M acetic acid using a dialysis
membrane with a nominal cut-off molecular weight of 1000. The
retentate is lyophilized. Decitraconylation is achieved in formic
acid pH 3.5 for 72 h, followed by renewed lyophilization. The
t-butyl ester is cleaved by dissolution in trifluoroacetic acid.
After evaporation of the trifluoroacetic acid, the protein is
dissolved in 1 M acetic acid and fractionated by chromatography on
Sephadex G-50 in 1 M acetic acid. The major protein peak is
collected and lyophilized. Then the material is once more
fractionated on the same column. The fractionated protein is
lyophilized. Ion-spray mass spectrometry shows the correct
molecular weight for the expected Compound F. (FIG. 5B). Compound F
comprises a therapeutic moiety (an insulin molecule) attached to a
cholic acid derivative linker, which is attached to a diagnostic
moiety (a metal chelator).
B. Synthesis of .sup.111In-labelled Compound F (Compound
.sup.111In--F)
To a 5-mL glass vial fitted with a 400 .mu.L autosampler insert is
added 100 .mu.L of a 1 mg/mL solution of Compound F dissolved in a
9:1 mixture of 0.2 M NaOAc buffer (pH 4.8) and DMF.
.sup.111InCl.sub.3 [1.5 mCi] in 0.05N HCl is added, the reaction
vial is crimp-sealed, and the solution is heated at 45.degree. C.
for 30 minutes. After being cooled to room temperature, an aliquot
of labeling mixture is purified by reversed phase UPLC on a Vydac
C-18 Peptide and Protein column [4.6.times.250 mm; pore size: 5
micron; flow rate: 1.5 mL/min at 30.degree. C.] using an
aqueous/organic gradient of 0.1% trifluoroacetic acid in
H.sub.2O/0.085% trifluoroacetic acid in acetonitrile. The peak
corresponding to .sup.111In-labeled Compound F is collected into
0.5 mL of 50 mM citrate buffer, pH 5.7 containing 0.2% Human Serum
Albumin, followed by removal of acetonitrile at reduced pressure
using a Savant Speed Vacuum apparatus.
Example VIII
FIGS. 6A-B
Synthesis of L62
Summary: As shown in FIGS. 6A-B, L62 was prepared using the
following steps: Hydrolysis of
(3.beta.,5.beta.)-3-aminocholan-24-oic acid methyl ester A with
NaOH gave the corresponding acid B, which was then reacted with
Fmoc-Cl to give intermediate C. Rink amide resin functionalised
with the octapeptide Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH.sub.2
(BBN[7-14] [SEQ ID NO:1]) was sequentially reacted with C,
Fmoc-glycine and DOTA tri-t-butyl ester. After cleavage and
deprotection with reagent B the crude was purified by preparative
HPLC to give L62. Overall yield: 2.5%. More details are provided
below:
A. Rink Amide Resin Functionalised with Bombesin[7-14], (A)
In a solid phase peptide synthesis vessel (see enclosure No. 1)
Fmoc-aminoacid (24 mmol), N-hydroxybenzotriazole (HOBt) (3.67 g; 24
mmol), and N,N'-diisopropylcarbodiimide (DIC) (3.75 mL; 24 mmol)
were added sequentially to a suspension of Rink amide NovaGel.TM.
resin (10 g; 6.0 mmol) A in DMF (45 mL). The mixture was shaken for
3 h at room temperature using a bench top shaker, then the solution
was emptied and the resin was washed with DMF (5.times.45 mL). The
resin was shaken with 25% piperidine in DMF (45 mL) for 4 min, the
solution was emptied and fresh 25% piperidine in DMF (45 mL) was
added. The suspension was shaken for 10 min, then the solution was
emptied and the resin was washed with DMF (5.times.45 mL).
This procedure was applied sequentially for the following amino
acids: N-.alpha.-Fmoc-L-methionine, N-.alpha.-Fmoc-L-leucine,
N-.alpha.-Fmoc-N-im-trityl-L-histidine, N-.alpha.-Fmoc-glycine,
N-.alpha.-Fmoc-L-valine, N-.alpha.-Fmoc-L-alanine,
N-.alpha.-Fmoc-N-in-Boc-L-tryptophan.
In the last coupling reaction
N-.alpha.-Fmoc-N-.gamma.-trityl-L-glutamine (14.6 g; 24 mmol), HOBt
(3.67 g; 24 mmol), and DIC (3.75 mL; 24 mmol) were added to the
resin in DMF (45 mL). The mixture was shaken for 3 h at room
temperature, the solution was emptied and the resin was washed with
DMF (5.times.45 mL), CH.sub.2Cl.sub.2 (5.times.45 mL) and vacuum
dried.
B. Preparation of Intermediates B and C:
1. Synthesis of (3.beta.,5.beta.)-3-Aminocholan-24-oic acid (B)
A 1 M solution of NaOH (16.6 mL; 16.6 mmol) was added dropwise to a
solution of (3.beta.,5.beta.)-3-aminocholan-24-oic acid methyl
ester (5.0 g; 12.8 mmol) in MeOH (65 mL) at 45.degree. C. After 3 h
stirring at 45.degree. C., the mixture was concentrated to 25 mL
and H.sub.2O (40 mL) and 1 M HCl (22 mL) were added. The
precipitated solid was filtered, washed with H.sub.2O (2.times.50
mL) and vacuum dried to give B as a white solid (5.0 g; 13.3 mmol).
Yield 80%.
2. Synthesis of
(3.beta.,5.beta.)-3-(9H-Fluoren-9-ylmethoxy)aminocholan-24-oic Acid
(C)
A solution of 9-fluorenylmethoxycarbonyl chloride (0.76 g; 2.93
mmol) in 1,4-dioxane (9 mL) was added dropwise to a suspension of
(3.beta.,5.beta.)-3-aminocholan-24-oic acid B (1.0 g; 2.66 mmol) in
10% aq. Na.sub.2CO.sub.3 (16 mL) and 1,4-dioxane (9 mL) stirred at
0.degree. C. After 6 h stirring at room temperature H.sub.2O (90
mL) was added, the aqueous phase washed with Et.sub.2O (2.times.90
mL) and then 2 M HCl (15 mL) was added (final pH: 1.5). The aqueous
phase was extracted with EtOAc (2.times.100 mL), the organic phase
dried over Na.sub.2SO.sub.4 and evaporated. The crude was purified
by flash chromatography to give C as a white solid (1.2 g; 2.0
mmol). Yield 69%.
C. Synthesis of L62
(N-[(3.beta.,5.beta.)-3-[[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraaza-
cyclododec-1-yl]acetyl]amino]
acetyl]amino]-cholan-24-yl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-gl-
ycyl-L-histidyl-L-leucyl-L-methioninamide)
Resin A (0.5 g; 0.3 mmol) was shaken in a solid phase peptide
synthesis vessel with 50% morpholine in DMA (7 mL) for 10 min, the
solution was emptied and fresh 50% morpholine in DMA (7 mL) was
added. The suspension was shaken for 20 min then the solution was
emptied and the resin washed with DMA (5.times.7 mL).
(3.beta.,5.beta.)-3-(9H-Fluoren-9-ylmethoxy)aminocholan-24-oic acid
C (0.72 g; 1.2 mmol), N-hydroxybenzotriazole (HOBT) (0.18 g; 1.2
mmol), N,N'-diisopropylcarbodiimide (DIC) (0.19 mL; 1.2 mmol) and
DMA (7 mL) were added to the resin, the mixture shaken for 24 h at
room temperature, and the solution was emptied and the resin washed
with DMA (5.times.7 mL). The resin was then shaken with 50%
morpholine in DMA (7 mL) for 10 min, the solution was emptied,
fresh 50% morpholine in DMA (7 mL) was added and the mixture shaken
for another 20 min. The solution was emptied and the resin washed
with DMA (5.times.7 mL). N-.alpha.-Fmoc-glycine (0.79 g; 1.2 mmol),
HOBT (0.18 g; 1.2 mmol), DIC (0.19 mL: 1.2 mmol) and DMA (7 mL)
were added to the resin. The mixture was shaken for 3 h at room
temperature, the solution was emptied and the resin washed with DMA
(5.times.7 mL). The resin was then shaken with 50% morpholine in
DMA (7 mL) for 10 min, the solution was emptied, fresh 50%
morpholine in DMA (7 mL) was added and the mixture shaken for
another 20 min. The solution was emptied and the resin washed with
DMA (5.times.7 mL) followed by addition of
1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid
tris(1,1-dimethylethyl) ester adduct with NaCl (0.79 g; 1.2 mmol),
HOBT (0.18 g; 1.2 mmol), DIC (0.19 mL: 1.2 mmol), DIEA (0.40 mL;
2.4 mmol) and DMA (7 mL) to the resin. The mixture was shaken for
24 h at room temperature, the solution was emptied and the resin
washed with DMA (5.times.7 mL), CH.sub.2Cl.sub.2 (5.times.7 mL) and
vacuum dried. The resin was shaken in a flask with reagent B (25
mL) for 4.5 h. The resin was filtered and the solution was
evaporated under reduced pressure to afford an oily crude that was
triturated with Et.sub.2O (20 mL). The precipitate was collected by
centrifugation and washed with Et.sub.2O (3.times.20 mL), then
analysed by HPLC and purified by preparative HPLC. The fractions
containing the product were lyophilised to give L62 (6.6 mg;
3.8.times.10.sup.-3 mmol) as a white solid. Yield 4.5%.
Example IX
FIGS. 7A-E
Synthesis of L67
Summary: Hydrolysis of
(3.beta.,5.beta.)-3-amino-12-oxocholan-24-oic acid methyl ester A
with NaOH gave the corresponding acid B, which was then reacted
with Fmoc-Glycine to give intermediate C. Rink amide resin
functionalised with the octapeptide
Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH.sub.2 (BBN[7-14] [SEQ ID NO:1])
was sequentially reacted with C, and DOTA tri-t-butyl ester. After
cleavage and deprotection with reagent B the crude was purified by
preparative HPLC to give L67. Overall yield: 5.2%.
A. Synthesis (3.beta.,5.beta.-3-Amino-12-oxocholan-24-oic acid,
(B)(FIG. 7A)
A 1 M solution of NaOH (6.6 mL; 6.6 mmol) was added dropwise to a
solution of (3.beta.,5.beta.)-3-amino-12-oxocholan-24-oic acid
methyl ester A (2.1 g; 5.1 mmol) in MeOH (15 mL) at 45.degree. C.
After 3 h stirring at 45.degree. C., the mixture was concentrated
to 25 mL then H.sub.2O (25 mL) and 1 M HCl (8 mL) were added. The
precipitated solid was filtered, washed with H.sub.2O (2.times.30
mL) and vacuum dried to give B as a white solid (1.7 g; 4.4 mmol).
Yield 88%.
B. Synthesis of
(3.beta.,5.beta.)-3-[[(9H-Fluoren-9-ylmethoxy)amino]acetyl]amino-12-oxoch-
olan-24-oic acid (C)(FIG. 7A)
Tributylamine (0.7 mL; 3.1 mmol) was added dropwise to a solution
of N-.alpha.-Fmoc-glycine (0.9 g; 3.1 mmol) in THF (25 mL) stirred
at 0.degree. C. Isobutyl chloroformate (0.4 mL; 3.1 mmol) was
subsequently added and, after 10 min, a suspension of tributylamine
(0.6 mL; 2.6 mmol) and
(3.beta.,5.beta.)-3-amino-12-oxocholan-24-oic acid B (1.0 g; 2.6
mmol) in DMF (30 mL) was added dropwise, over 1 h, into the cooled
solution. The mixture was allowed to warm up and after 6 h the
solution was concentrated to 40 mL, then H.sub.2O (50 mL) and 1 N
HCl (10 mL) were added (final pH: 1.5). The precipitated solid was
filtered, washed with H.sub.2O (2.times.50 mL), vacuum dried and
purified by flash chromatography to give C as a white solid (1.1 g;
1.7 mmol). Yield 66%.
C. Synthesis of L67
(N-[3.beta.,5.beta.)-3-[[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazac-
yclododec-1-yl]acetyl]amino]acetyl]amino]-12,24-dioxocholan-24-yl]-L-gluta-
minyl-L-trwptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methionin-
amide) (FIGS. 7B and 7E).
Resin D (0.5 g; 0.3 mmol) was shaken in a solid phase peptide
synthesis vessel with 50% morpholine in DMA (7 mL) for 10 min, the
solution was emptied and fresh 50% morpholine in DMA (7 mL) was
added. The suspension was stirred for 20 min then the solution was
emptied and the resin was washed with DMA (5.times.7 mL).
(3.beta.,5.beta.)-3-[[(9H-Fluoren-9-ylmethoxy)amino]acetyl]amino]-12-oxoc-
holan-24-oic acid C (0.80 g; 1.2 mmol), N-hydroxybenzotriazole
(HOBT) (0.18 g; 1.2 mmol), N,N'-diisopropylcarbodiimide (DIC) (0.19
mL; 1.2 mmol) and DMA (7 mL) were added to the resin, the mixture
was shaken for 24 h at room temperature, the solution was emptied
and the resin was washed with DMA (5.times.7 mL). The resin was
shaken with 50% morpholine in DMA (7 mL) for 10 min, the solution
was emptied, fresh 50% morpholine in DMA (7 mL) was added and the
mixture was shaken for 20 min. The solution was emptied and the
resin washed with DMA (5.times.7 mL).
1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid
tris(1,1-dimethylethyl) ester adduct with NaCl (0.79 g; 1.2 mmol),
HOBT (0.18 g; 1.2 mmol), DIC (0.19 mL: 1.2 mmol), DEEA (0.40 mL;
2.4 mmol) and DMA (7 mL) were added to the resin. The mixture was
shaken for 24 h at room temperature, the solution was emptied and
the resin was washed with DMA (5.times.7 mL), CH.sub.2Cl.sub.2
(5.times.7 mL) and vacuum dried. The resin was shaken in a flask
with reagent B (25 mL) for 4.5 h. The resin was filtered and the
solution was evaporated under reduced pressure to afford an oily
crude that was triturated with Et.sub.2O (20 mL). L67 comprises a
diagnostic moiety (a metal chelator) attached to a cholic acid
derivative linker, which is attached to a targeting peptide
(BBN[7-14]).
Example X
FIGS. 8A-H
Synthesis of L63 and L64
Summary: Hydrolysis of
(3.beta.,5.beta.,7.alpha.,12.alpha.)-3-amino-7,12-dihydroxycholan-24-oic
acid methyl ester 1b with NaOH gave the intermediate 2b, which was
then reacted with Fmoc-glycine to give 3b. Rink amide resin
functionalised with the octapeptide
Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH.sub.2 (BBN[7-14] [SEQ ID NO:1])
was reacted with 3b and then with DOTA tri-t-butyl ester. After
cleavage and deprotection with reagent B the crude was purified by
preparative HPLC to give L64. The same procedure was repeated
starting from intermediate 2a, already available, to give L63.
Overall yields: 9 and 4%, respectively.
A. Synthesis of (3.beta.,5.beta.,7.alpha.,
12.alpha.)-3-Amino-7,12-dihydroxycholan-24-oic acid. (2b)(FIG.
8A)
A 1 M solution of NaOH (130 mL; 0.13 mol) was added dropwise to a
solution of
(3.beta.,5.beta.,7.alpha.,12.alpha.)-3-amino-7,12-dihydroxycholan-24-oic
acid methyl ester 1b (42.1 g; 0.10 mol) in MeOH (300 mL) heated at
45.degree. C. After 3 h stirring at 45.degree. C., the mixture was
concentrated to 150 mL and H.sub.2O (350 mL) was added. After
extraction with CH.sub.2Cl.sub.2 (2.times.100 mL) the aqueous
solution was concentrated to 200 mL and 1 M HCl (150 mL) was added.
The precipitated solid was filtered, washed with H.sub.2O
(2.times.100 mL) and vacuum dried to give 2b as a white solid (34.8
g; 0.08 mol). Yield 80%.
B. Synthesis of (3.beta.,
5.beta.,12.alpha.)-3-[[(9H-Fluoren-9-ylmethoxy)amino]acetyl]amino-12-hydr-
oxycholan-24-oic acid. (3a) (FIG. 8A)
Tributylamine (4.8 mL; 20.2 mmol) was added dropwise to a solution
of N-.alpha.-Fmoc-glycine (6.0 g; 20.2 mmol) in TIE (120 mL)
stirred at 0.degree. C. Isobutyl chloroformate (2.6 mL; 20.2 mmol)
was subsequently added and, after 10 min, a suspension of
tributylamine (3.9 mL; 16.8 mmol) and
(3.beta.,5.beta.,12.alpha.)-3-amino-12-hydroxycholan-24-oic acid 2a
(6.6 g; 16.8 mmol) in DMF (120 mL) was added dropwise, over 1 h,
into the cooled solution. The mixture was allowed to warm up and
after 6 h the solution was concentrated to 150 mL, then H.sub.2O
(250 mL) and 1 N HCl (40 mL) were added (final pH: 1.5). The
precipitated solid was filtered, washed with H.sub.2O (2.times.100
mL), vacuum dried and purified by flash chromatography to give 3a
as a white solid (3.5 g; 5.2 mmol). Yield 31%.
C. Synthesis of
(3.beta.,5.beta.,7.alpha.,12.alpha.)-3-[[(9H-Fluoren-9-ylmethoxy)amino]ac-
etyl]amino-7,12-dihydroxycholan-24-oic acid, (3b) (FIG. 8B)
Tributylamine (3.2 mL; 13.5 mmol) was added dropwise to a solution
of N-.alpha.-Fmoc-glycine (4.0 g; 13.5 mmol) in THF (80 mL) stirred
at 0.degree. C. Isobutyl chloroformate (1.7 mL; 13.5 mmol) was
subsequently added and, after 10 min, a suspension of tributylamine
(2.6 mL; 11.2 mmol) and
(3.beta.,5.beta.,7.alpha.,12.alpha.)-3-amino-7,12-dihydroxychol-
an-24-oic acid 3a (4.5 g; 11.2 mmol) in DMF (80 mL) was added
dropwise, over 1 h, into the cooled solution. The mixture was
allowed to warm up and after 6 h the solution was concentrated to
120 mL, then H.sub.2O (180 mL) and 1 N HCl (30 mL) were added
(final pH: 1.5). The precipitated solid was filtered, washed with
H.sub.2O (2.times.100 mL), vacuum dried and purified by flash
chromatography to give 3a as a white solid (1.9 g; 2.8 mmol). Yield
25%. In an alternative method,
(3.beta.,5.beta.,7.alpha.,12.alpha.)-3-[[(9H-Fluoren-9-ylmethoxy)amino]ac-
etyl]amino-7,12-dihydroxycholan-24-oic acid, (3b) can be prepared
as follows: N-Hydroxysuccinimide (1.70 g, 14.77 mmol) and DIC (1.87
g, 14.77 mmol) were added sequentially to a stirred solution of
Fmoc-Gly-OH (4.0 g, 13.45 mmol) in dichloromethane (15 mL); the
resulting mixture was stirred at room temperature for 4 h. The
N,N'-diisopropylurea formed was removed by filtration and the solid
was washed with ether (20 mL). The volatiles were removed and the
solid Fmoc-Gly-succinimidyl ester formed was washed with ether
(3.times.20 mL). Fmoc-Gly-succinimidyl ester was then redissolved
in dry DMF (15 mL) and 3-aminodeoxycholic acid (5.21 g, 12.78 mmol)
was added to the clear solution. The reaction mixture was stirred
at room temperature for 4 h, water (200 mL) was added and the
precipitated solid was filtered, washed with water, dried and
purified by silica gel chromatography (TLC (silica): (R.sub.f:
0.50, silica gel, CH.sub.2Cl.sub.2/CH.sub.3OH, 9:1) (eluant:
CH.sub.2Cl.sub.2/CH.sub.3OH (9:1) to give
(3.beta.,5.beta.,7.alpha.,12.alpha.)-3-[[(9H-Fluoren-9-ylmethoxy)amino]ac-
etyl]amino-7,12-dihydroxycholan-24-oic acid, (3b) as a colorless
solid. Yield: 7.46 g (85%).
D. Synthesis of L63 (N-[(3.beta.,5.beta.,
12.alpha.)-3-[[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec--
1-yl]acetyl]amino]acetyl]amino]-12-hydroxy-24-oxocholan-24-yl]-L-glutaminy-
l-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidvl-L-leucvl-L-methioninamid-
e) (FIGS. 8B and 8G)
Resin A (0.5 g; 0.3 mmol) was shaken in a solid phase peptide
synthesis vessel with 50% morpholine in DMA (7 mL) for 10 min, the
solution was emptied and fresh 50% morpholine in DMA (7 mL) was
added. The suspension was stirred for 20 min then the solution was
emptied and the resin washed with DMA (5.times.7 mL).
(3.beta.,5.beta.,12.alpha.)-3-[[(9H-Fluoren-9-ylmethoxy)amino]acetyl]amin-
o-12-hydroxycholan-24-oic acid 3a (0.82 g; 1.2 mmol),
N-hydroxybenzotriazole (HOBI) (0.18 g; 1.2 mmol),
N,N'-diisopropylcarbodiimide (DIC) (0.19 mL; 1.2 mmol) and DMA (7
mL) were added to the resin, the mixture was shaken for 24 h at
room temperature, the solution was emptied and the resin was washed
with DMA (5.times.7 mL). The resin was then shaken with 50%
morpholine in DMA (7 mL) for 10 min, the solution was emptied,
fresh 50% morpholine in DMA (7 mL) was added and the mixture was
shaken for 20 min. The solution was emptied and the resin washed
with DMA (5.times.7 mL).
1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid
tris(1,1-dimethylethyl) ester adduct with NaCl (0.79 g; 1.2 mmol),
HOBT (0.18 g; 1.2 mmol), DIC (0.19 mL: 1.2 mmol), DIEA (0.40 mL;
2.4 mmol) and DMA (7 mL) were added to the resin. The mixture was
shaken for 24 h at room temperature, the solution was emptied and
the resin washed with DMA (5.times.7 mL), CH.sub.2Cl.sub.2
(5.times.7 mL) and vacuum dried. The resin was shaken in a flask
with reagent B (25 mL) for 4 h. The resin was filtered and the
solution was evaporated under reduced pressure to afford an oily
crude that after treatment with Et.sub.2O (5 mL) gave a
precipitate. The precipitate was collected by centrifugation and
washed with Et.sub.2O (5.times.5 mL), then analysed and purified by
HPLC. The fractions containing the product were lyophilised to give
L63 as a white fluffy solid (12.8 mg; 0.0073 mmol). Yield 9%. L63
comprises a diagnostic moiety (a metal chelator) attached to a
cholic acid derivative linker, which is attached to a targeting
peptide (BBN[7-14]).
E. Synthesis of L64
(N-[(3.beta.,5.beta.,7.alpha.,12.alpha.)-3-[[[[[4,7,10-Tris(carboxymethyl-
)-1,4,7,10-tetraazacyclododec-1-yl]
acetyl]amino]acetyl]amino]-7,12-dihydroxy-24-oxocholan-24-yl]-L-glutaminy-
l-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamid-
e) (FIGS. 8C and 8H)
Resin A (0.5 g; 0.3 mmol) was shaken in a solid phase peptide
synthesis vessel with 50% morpholine in DMA (7 mL) for 10 min, the
solution was emptied and fresh 50% morpholine in DMA (7 mL) was
added. The suspension was stirred for 20 min, the solution was
emptied and the resin was washed with DMA (5.times.7 mL).
(3.beta.,5.beta.,7.alpha.,12.alpha.)-3-[[(9H-Fluoren-9-ylmethoxy)amino]ac-
etyl]amino-7,12-dihydroxycholan-24-oic acid 3b (0.81 g; 1.2 mmol),
HOBT (0.18 g; 1.2 mmol), DIC (0.19 mL; 1.2 mmol) and DMA (7 mL)
were added to the resin, the mixture shaken for 24 h at room
temperature, the solution was emptied and the resin washed with DMA
(5.times.7 mL). The resin was shaken with 50% morpholine in DMA (7
mL) for 10 min, the solution was emptied, fresh 50% morpholine in
DMA (7 mL) was added and the mixture was shaken for 20 min. The
solution was emptied and the resin was washed with DMA (5.times.7
mL). 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid
tris(1,1-dimethylethyl) ester adduct with NaCl (0.79 g; 1.2 mmol),
HOBT (0.18 g; 1.2 mmol), DIC (0.19 mL: 1.2 mmol), DIEA (0.40 mL;
2.4 mmol) and DMA (7 mL) were added to the resin. The mixture was
shaken for 24 h at room temperature, the solution was emptied and
the resin washed with DMA (5.times.7 mL), CH.sub.2Cl.sub.2
(5.times.7 mL) and vacuum dried. The resin was shaken in a flask
with reagent B (25 mL) for 4 h. The resin was filtered and the
solution was evaporated under reduced pressure to afford an oily
crude that was triturated with Et.sub.2O (5 mL). The precipitate
was collected by centrifugation and washed with Et.sub.2O
(5.times.5 mL). Then it was dissolved in H.sub.2O (20 mL), and
Na.sub.2CO.sub.3 (0.10 g; 0.70 mmol) was added; the resulting
mixture was stirred 4 h at room temperature. This solution was
purified by HPLC, the fractions containing the product lyophilised
to give L64 as a white fluffy solid (3.6 mg; 0.0021 mmol). Yield
4%. L64 comprises a diagnostic moiety (a metal chelator) attached
to a cholic acid derivative linker, which is attached to a
targeting peptide (BBN[7-14]).
Example XI
Preparation of .sup.177Lu-L64 for Cell Binding and Biodistribution
Studies
This compound was synthesized by incubating 10 .mu.g L64 ligand (10
.mu.L of a 1 mg/mL solution in water), 100 .mu.L ammonium acetate
buffer (0.2M, pH 5.2) and .about.1-2 mCi of .sup.177LuCl.sub.3 in
0.05N HCl (MURR) at 90.degree. C. for 15 min. Free .sup.177Lu was
scavenged by adding 20 .mu.L of a 1% Na.sub.2EDTA.2H.sub.2O
(Aldrich) solution in water. The resulting radiochemical purity
(RCP) was .about.95%. The radiolabeled product was separated from
unlabeled ligand and other impurities by HPLC, using a YMC Basic C8
column [4.6.times.150 mm], a column temperature of 30.degree. C.
and a flow rate of 1 mL/min. with a gradient of 68% A/32% B to 66%
A/34% B over 30 min., where A is citrate buffer (0.02M, pH 3.0),
and B is 80% CH.sub.3CN/20% CH.sub.3OH. The isolated compound had
an RCP of .about.100% and an HPLC retention time of 23.4
minutes.
Samples for biodistribution and cell binding studies were prepared
by collecting the desired HPLC peak into 1000 .mu.L of citrate
buffer (0.05 M, pH 5.3, containing 1% ascorbic acid, and 0.1% HSA).
The organic eleunt in the collected eluate was removed by
centrifugal concentration for 30 min. For cell binding studies, the
purified sample was diluted with cell-binding media to a
concentration of 1.5 .mu.Ci/mL within 30 minutes of the in vitro
study. For biodistribution studies, the sample was diluted with
citrate buffer (0.05 M, pH 5.3, containing 1% sodium ascorbic acid
and 0.1% HSA) to a final concentration of 50 .mu.Ci/mL within 30
minutes of the in vivo study.
Example XII
Preparation of .sup.177Lu-L64 for Radiotherapy Studies
This compound was synthesized by incubating 70 .mu.g L64 ligand (70
.mu.L of a 1 mg/mL solution in water), 200 .mu.L ammonium acetate
buffer (0.2M, pH 5.2) and .about.30-40 mCi of .sup.177LuCl.sub.3 in
0.05N HCl (MURR) at 85.degree. C. for 10 min. After cooling to room
temperature, free .sup.177Lu was scavenged by adding 20 .mu.L of a
2% Na.sub.2EDTA.2H.sub.2O (Aldrich) solution in water. The
resulting radiochemical purity (RCP) was -95%. The radiolabeled
product was separated from unlabeled ligand and other impurities by
HPLC, using a 300VHP Anion Exchange column (7.5.times.50 mm)
(Vydac) that was sequentially eluted at a flow rate of 1 mL/min
with water, 50% acetonitrile/water and then 1 g/L aqueous ammonium
acetate solution. The desired compound was eluted from the column
with 50% CH.sub.3CN and mixed with .about.1 mL of citrate buffer
(0.05 M, pH 5.3) containing 5% ascorbic acid, 0.2% HSA, and 0.9%
(v:v) benzyl alcohol. The organic part of the isolated fraction was
removed by spin vacuum for 40 min, and the concentrated solution
(.about.20-25 mCi) was adjusted within 30 minutes of the in vivo
study to a concentration of 7.5 mCi/mL using citrate buffer (0.05
M, pH 5.3) containing 5% ascorbic acid, 0.2% HSA, and 0.9% (v:v)
benzyl alcohol. The resulting compound had an RCP of >95%.
Example XIII
Preparation of .sup.111In-L64
This compound was synthesized by incubating 10 .mu.g L64 ligand (5
.mu.L of a 2 mg/mL solution in 0.01 N HCl), 60 .mu.L ethanol, 1.12
mCi of .sup.111InCl.sub.3 in 0.05N HCl (80 .mu.L) and 155 .mu.L
sodium acetate buffer (0.5M, pH 4.5) at 85.degree. C. for 30 min.
Free .sup.111In was scavenged by adding 20 .mu.L of a 1%
Na.sub.2EDTA.2H.sub.2O (Aldrich) solution in water. The resulting
radiochemical purity (RCP) was 87%. The radiolabeled product was
separated from unlabeled ligand and other impurities by HPLC, using
a Vydac C18 columns [4,6.times.250 mm], a column temperature of
50.degree. C. and a flow rate of 1.5 mL/min. with a gradient of 75%
A/25% B to 65% A/35% B over 20 min where A is 0.1% TFA in water, B
is 0.085% TFA in acetonitrile. With this system, the retention time
for .sup.111n-L64 is 15.7 min. The isolated compound had an RCP of
96.7%.
Example XIV
Preparation of .sup.177Lu-L63
A stock solution of peptide was prepared by dissolving L63 ligand
(prepared as described in US Application Publication No.
2002/0054855 and WO 02/87637, both incorporated by reference) in
0.01 N HCl to a concentration of 1 mg/mL. .sup.177Lu-L63 was
prepared by mixing the following reagents in the order shown.
TABLE-US-00004 0.2 M NH.sub.4OAc, pH 6.8 100 .mu.l Peptide stock, 1
mg/mL, in 0.01 N HCl 5 .mu.l .sup.177LuCl.sub.3 (MURR) in 0.05 M
HCl 1.2 .mu.l (1.4 mCi)
The reaction mixture was incubated at 85.degree. C. for 10 min.
After cooling down to room temperature in a water bath, 20 .mu.l of
a 1% EDTA solution and 20 .mu.l of EtOH were added. The compound
was analyzed by HPLC using a C18 column (VYDAC Cat # 218TP54) that
was eluted at flow rate of 1 mL/min with a gradient of 30-34% B
over 20 min (where solvent is A. 0.1% TFA/H.sub.2O and B is 0.1%
TFA/CH.sub.3CN). The .sup.177Lu-L63 that formed had an RCP of 97.8%
and a retention time of 14.2 min on this system.
Example XV
Relaxivity and HSA Binding of Gd-L64--FIGS. 11A-C
Gd-L64 displays a relaxivity in HEPES of about 10.45
mM.sup.-1s.sup.-1, which is consistent with the expected value
based on moledular weight.
As shown in FIG. 11A, titration of a HEPES solution of Gd-L64 with
HSA shows relatively strong binding
(K.sub.a=1.2.times.10.sup.4M.sup.-1; R.sub.b=20 mM.sup.-1s.sup.-1).
Fitting the experimental curve yields a K.sub.a value of
1.2.times.10.sup.4 M.sup.-1 and a relaxivity of the fully bound
species of about 20 mM.sup.-1s.sup.-1.
As shown in FIG. 11B, binding of Gd-L64 to HSA is confirmed by
comparing NMRD profiles in HEPES and in serum, where the
characteristic peak of bound species at frequencies of 10-30 MHz is
seen in the presence of HAS. These results indicate that compounds
containing linkers of the invention bind to HAS despite the absence
of a free carboxylic acid at the 24 position.
Example XVI
In Vivo Pharmacokinetic Studies
A. Tracer Dose Biodistribution: Low dose pharmacokinetic studies
(e.g., biodistribution studies) were performed using the
below-identified compounds of the invention and L 134, a control,
in xenografted, PC3 tumor-bearing mice ([Ncr]-Foxn1<nu>).
L134 is DO3A monoatnide-aminooctanyl-BBN[7-14]. In all studies,
mice were administered 100 .mu.L of .sup.177Lu-labelled test
compound at 200 .mu.Ci/kg, i.v., with a residence time of 1 and 24
h per group (n=3-4). Tissues were analyzed in an LKB 1282
CompuGamma counter with appropriate standards.
TABLE-US-00005 TABLE 2 Pharmacokinetic comparison at 1 and 24 h in
PC3 tumor-bearing nude mice (200 .mu.Ci/kg; values as % ID/g) of
Lu-177 labelled compounds of the invention compared to control L134
L63 L64 Tissue 1 hr 24 hr 1 hr 24 hr 1 hr 24 hr Blood 0.44 0.03
7.54 0.05 1.87 0.02 Liver 0.38 0.04 12.15 0.20 2.89 0.21 Kidneys
7.65 1.03 7.22 0.84 10.95 1.45 Tumor 3.66 1.52 9.49 2.27 9.83 3.60
Pancreas 28.60 1.01 54.04 1.62 77.78 6.56
Whereas the distribution of radioactivity in the blood, liver and
kidneys after injection of L64 is similar to that of the control
compound, L134, the uptake in the tumor is much higher at 1 and 24
h for L64. L63 also shows high tumour uptake although with
increased blood and liver values at early times. Uptake in the
mouse pancreas, a normal organ known to have GRP receptors is much
higher for L64 and L63 than for L134.
Example XVII
Radiotherapy Studies--FIGS. 12A-B
A. Short Term Efficacy Studies: Radiotherapy studies were performed
using the PC3 tumor-bearing nude mouse model. Lu-177 labelled
compounds of the invention L64, L63 and the treatment control
compound L134 were compared to an untreated control group. (n=12
for each treatment group and n=36 for the untreated control group).
For the first study, mice were administered 100 .mu.L of
.sup.177Lu-labelled compound of the invention at 30 mCi/kg, i.v, or
s.c. under sterile conditions. The subjects were housed in a
barrier environment for up to 30 days. Body weight and tumor size
(by caliper measurement) were collected on each subject 3 times per
week for the duration of the study. Criteria for early termination
included: death; loss of total body weight (TBW) equal to or
greater than 20%; tumor size equal to or greater than 2 cm.sup.3.
Results are displayed in FIG. 12A. These results show that animals
treated with L64 or L63 have increased survival over the control
animals given no treatment and over those animals given the same
dose of L134. A repeat study was performed with L64 using the same
dose as before but using more animals per compound (n=46) and
following them for longer. The results of the repeat study are
displayed in FIG. 12B. Relative to the same controls as before
(n=36), L64 treatment gave significantly increased survival
(p<0.0001).
Example XVIII
Synthesis of LHRH Peptides
Linear peptides were synthesized using an Applied Biosystems
Peptide Synthesizer, model 433A. Fmoc-Pal-Peg-PS resin was used for
peptide synthesis, applying an orthogonal Fmoc strategy. The amino
side chains were protected with trityl- (for His), t-butyl-(for Ser
and Tyr), boc-(for Trp), methyltrityl-(for D-Lys) and Pmc- (for
Arg) groups. Cleavage of the peptides and workup were performed as
follows: Treatment of the resin loaded with the peptide of interest
with 88% trifluoroacetic acid (TFA)/5% ophenol/5% water/2%
triisopropylsilane (TIS) (reagent `B`) at room temperature for 4 h
and subsequent evaporation and precipitation in diethyl ether
provided crude peptide. The off-colored white solid was then
dissolved in water, filtered and purified by HPLC using a Shimadzu
System with a reversed phase C-18 column (YMC-Pack ODS/A column)
employing water and acetonitrile with 0.1% TFA as eluents.
A. Synthesis of Fmoc-Gly-3-Aminodeoxycholic Acid
(3.beta.,5.beta.,7.alpha.,12.alpha.)-3
[[9H-Fluoren-9-ylmethoxy)amino]acetyl]amino-7-12-dihydroxycholan-24-oic
acid
To a solution of Fmoc-Gly-OH (4.0 g, 13.45 mmol) in dichloromethane
(15 mL) was added N-hydroxysuccinimide (1.70 g, 14.77-mmol)
followed by DIC (diisopropyl-carbodiimide) (1.87 g, 14.77 mmol) and
stirred at room temperature for 4 h. The urea formed was removed by
filtration and washed the solid with ether (20 mL). The combined
filtrate was evaporated on a rotary evaporator to remove the
solvent and the solid, Fmoc-Gly-succinimidyl ester, formed was
washed with ether (3.times.20 mL). Fmoc-Gly-succinimidyl ester was
then redissolved in dry DMF (15 mL) and 3-aminodeoxycholic acid
(5.21 g, 12.78 mmol) was then added to the clear solution. After
stirring the reaction mixture at room temperature for 4 h, the
mixture was added to water (200 mL) and the precipitated solid was
filtered, washed with water, dried and purified by silica gel
chromatography (R.sub.f: 0.50, silica gel,
CH.sub.2Cl.sub.2/CH.sub.3OH, 9:1) (eluant:
CH.sub.2Cl.sub.2/CH.sub.3OH (9:1) to give
Fmoc-Gly-3-aminodeoxycholic acid as a colorless solid. Yield: 7.46
g (85%).
MS (API-ES, +ve ion): 710.2 (M+Na), 687.7 (M+H).
B.
Pyr-YH's-Trp-Ser-Tyr-DLys(DOTA-Gly-Adca3)-Leu-Arg-Pro-Gly-NH.sub.2
To a solution of Fmoc-Gly-3-aminodeoxycholic acid (20 mg, 0.029
mmol) in dichloromethane (0.2 mL) was added N-hydroxysuccinimide
(4.0 mg, 0.035 mmol) followed by DIC (diisopropylcarbodiimide) (5.0
mg, 0.039 mmol) and stirred at room temperature for 4 h. After
evaporation of the solvent, the solid was washed with diethyl ether
(5.times.1.0 mL) and dried. Solid of Fmoc-Gly-3-aminodeoxycholic
acid succinimidyl ester was then dissolved in dry DMF (0.5 mL) and
to the clear solution was added pure
H-Pyr-His-Trp-Ser-Tyr-DLys-Leu-Arg-Pro-Gly-NH.sub.2 (30 mg, 0.024
mmol) and diisopropylethylamine (7.5 mg, 0.058 mmol), and stirred
the reaction mixture at room temperature for 18 h. To reaction
mixture, piperidine (0.1 mL) was added and stirred for 20 min to
remove the Fmoc protecting group. Diluted with water, acidified the
solution to pH 4.0 with TFA and loaded onto a YMC-Pack ODS/A,
30.times.250 mm column. Eluted the column employing water and
acetonitrile with 0.1% TFA as eluents and collected the fractions
containing the required peptide (based on MS results) and
lyophilized to obtain pure
Pyr-His-Trp-Ser-Tyr-DLys(H-Gly-Adca3)-Leu-Arg-Pro-Gly-NH.sub.2 (15
mg, 41%) as a colorless fluffy solid. MS (API-ES, positive ion):
1700.4 (M+H), 851.5 [M+H]/2.
To the pre-activated solution of DOTA-tri-t-butyl ester (25.3 mg,
0.04 mmol), HBTU (15.2 mg, 0.04 mmol) and diisopropylethylamine (12
mg, 0.09 mmol) in dry DMF (0.2 mL) was added
Pyr-His-Trp-Ser-Tyr-DLys(H-Gly-Adca3)-Leu-Arg-Pro-Gly-NH.sub.2 (15
mg, 0.01 mmol)) and stirred the reaction mixture for 15 h. After
evaporation of the solvent under vacuum, the residue was treated
with TFA-anisole (95:5, 2 mL) for 5 h. After evaporation of the
volatiles on a rotary evaporator, the peptide was precipitated by
adding ether. The crude peptide was obtained by centrifugation,
washed with ether and isolated the required peptide
Pyr-His-Trp-Ser-Tyr-DLys(DOTA-Gly-Adca3)-Leu-Arg-Pro-Gly-NH.sub.2
as a colorless solid. Yield: 12.5 mg (68%).
MS (API-ES, positive ion): 2086.5 (M+H), 1044.3 [M+H]/2
Example XIX
Preparation of .sup.125I-labeled Compounds
Compounds of the invention can be labeld with a radioactive halogen
such as, for example, .sup.125I, .sup.131I or .sup.123I using
labeling and purification procedures that are known to those
skilled in the art or suitable modifications thereof. Typical
procedures for labeling with radioiodine (or other radioactive
halogens) using Bolton-Hunter Reagent, Lactoperoxidase, Iodogen and
Chloramine T reagents are described by A. E. Bolton, Comparative
Methods For The Radiolabeling Of Peptides, in "Methods in
Enzymology", 1986, Vol. 124, p. 18-29. For example, the compounds
of the present invention can be labeled with .sup.125I by reacting
the unlabeled compound in (e.g.) a small volume of borate buffer or
DMF that has been previously adjusted to pH 8.5-9.0 using, for
example, diisopropyl amine, with dried Bolton-Hunter reagent The
vial is shaken and then incubated on ice for about 30 minutes with
occasional shaking. After this time, the reaction can optionally be
quenched with a solution of (e.g.) glycine and then purified using,
for example, reversed phase HLPC to remove free labeled compound
from impurities and from unlabeled compound. Alternatively, the
compounds can be dissolved in phosphate buffer to a pH near 7,
iodide (as Na.sup.125I) can be added, and the solution treated with
a buffered solution of Chloramine T to effect iodination, then
treated with a reductant such as sodium meta bisulfite to quench
the reaction. Free I.sup.- can be removed by ion exchange
chromatography and unlabeled compound can be resolved from
radiolabeled compound using HPLC methods such as C8 or C18 reversed
phase chromatography.
Example XX
Synthesis of L69--FIGS. 13A and 13B
Summary: Reaction of
(3.beta.,5.beta.,7.alpha.,12.alpha.)-3-amino-7,12-dihydroxycholan-24-oic
acid A with Fmoc-Cl gave intermediate B. Rink amide resin
functionalised with the octapeptide
GlnTrpAlaValGlyHisLeuMetNH.sub.2 (BBN[7-14]) (A), was sequentially
reacted with B, Fmoc-8-amino-3,6-dioxaoctanoic acid and DOTA
tri-t-butyl ester. After cleavage and deprotection with reagent B
(2) the crude was purified by preparative HPLC to give L69. Overall
yield: 4.2%.
A.
(3.beta.,5.beta.,7.alpha.,12.alpha.)-3-(9H-Fluoren-9-ylmethoxy)amino-7,-
12-dihydroxycholan-24-oic acid, B (FIG. 13A)
A solution of 9-fluorenylmethoxycarbonyl chloride (1.4 g; 5.4 mmol)
in 1,4-dioxane (18 mL) was added dropwise to a suspension of
(3.beta.,5.beta.,7.alpha.,12.alpha.)-3-amino-7,12-dihydroxycholan-24-oic
acid A (2.0 g; 4.9 mmol) (3) in 10% aq. Na.sub.2CO.sub.3 (30 mL)
and 1,4-dioxane (18 mL) stirred at 0.degree. C. After 6 h stirring
at room temperature H.sub.2O (100 mL) was added, the aqueous phase
washed with Et.sub.2O (2.times.90 mL) and then 2 M HCl (15 mL) was
added (final pH: 1.5). The precipitated solid was filtered, washed
with H.sub.2O (3.times.100 mL), vacuum dried and then purified by
flash chromatography (4) to give B as a white solid (2.2 g; 3.5
mmol). Yield 71%.
B.
N-[(3.beta.,5.beta.,7.alpha.,12.alpha.)-3-[[[2-[2-[[[4,7,10-Tris(carbox-
ymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]ethoxy]ethoxy]acety-
l]amino]-7,12-dihydroxy-24-oxocholan-24-yl]-L-glutaminyl-L-tryptophyl-L-al-
anyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide, L69 (FIG.
13B)
Resin A (0.5 g; 0.3 mmol) (6) was shaken in a solid phase peptide
synthesis vessel with 50% morpholine in DMA (7 mL) for 10 min, the
solution flushed off and fresh 50% morpholine in DMA (7 mL) was
added. The suspension was stirred for another 20 min then the
solution was flushed off and the resin washed with DMA (5.times.7
mL).
(3.beta.,5.beta.,7.alpha.,12.alpha.)-3-(9H-Fluoren-9-ylmethoxy)amino-7,12-
-dihydroxycholan-24-oic acid B (0.75 g; 1.2 mmol),
N-hydroxybenzotriazole (HOBt) (0.18 g; 1.2 mmol),
N,N'-diisopropylcarbodiimide (DIC) (0.19 mL; 1.2 mmol) and DMA (7
mL) were added to the resin, the mixture shaken for 24 h at room
temperature, emptied and the resin washed with DMA (5.times.7 mL).
The resin was then shaken with 50% morpholine in DMA (7 mL) for 10
min, the solution emptied, fresh 50% morpholine in DMA (7 mL) was
added and the mixture shaken for another 20 min. The solution was
emptied and the resin washed with DMA (5.times.7 mL).
Fmoc-8-amino-3,6-dioxaoctanoic acid (0.79 g; 1.2 mmol) (7), HOBt
(0.18 g; 1.2 mmol), DIC (0.19 mL; 1.2 mmol) and DMA (7 mL) were
added to the resin. The mixture was shaken for 3 h at room
temperature, emptied and the resin washed with DMA (5.times.7 mL).
The resin was then shaken with 50% morpholine in DMA (7 mL) for 10
min, the solution flushed off, fresh 50% morpholine in DMA (7 mL)
was added and the mixture shaken for another 20 min. The solution
was flushed off and the resin washed with DMA (5.times.7 mL)
1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid
tris(1,1-dimethylethyl) ester adduct with NaCl (0.79 g; 1.2 mmol),
HOBt (0.18 g; 1.2 mmol), DIC (0.19 mL: 1.2 mmol),
N-ethyldiisopropylamine (0.40 mL; 2.4 mmol) and DMA (7 mL) were
added to the resin. The mixture was shaken for 24 h at room
temperature, flushed off and the resin washed with DMA (5.times.7
mL), CH.sub.2Cl.sub.2 (5.times.7 mL) and vacuum dried. The resin
was shaken in a flask with Reagent B (25 mL) (2) for 4.5 h. The
resin was filtered and the solution was evaporated under reduced
pressure to afford an oily crude that after treatment with
Et.sub.2O (20 mL) gave a precipitate. The precipitate was collected
by centrifugation and washed with Et.sub.2O (3.times.20 mL) to give
a solid (248 mg) which was analysed by HPLC. An amount of crude (50
mg) was purified by preparative HPLC. The fractions containing the
product were lyophilised to give L69 (6.5 mg; 3.5.times.10.sup.-3
mmol) (FIG. 13B) as a white solid. Yield 5.8%.
Throughout the foregoing description of the invention, various
patents, articles, and other publications have been cited or
referenced. The entire contents of each patent, article and other
publication is hereby incorporated by reference into the subject
application.
SEQUENCE LISTINGS
1
118PRTArtificialDescription of Artificial Sequence This peptide is
known as bombesin (i.e., BBN (7-14)) and binds to gastrin releasing
peptide (GRP) receptors. 1Gln Trp Ala Val Gly His Leu Met1 5
* * * * *